Biochem and Mol Bio

What are epitope tags?

Epitope tags are short peptide sequences which are chosen because high-affinity antibodies can be reliably produced in many different species. These are usually derived from viral genes, which explain their high immunoreactivity. Epitope tags include V5-tag, Myc-tag, HA-tag, Spot-tag and NE-tag. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.

What is guanidine hydrochloride?

Guanidine Hydrochloride is a strong chaotrope and one of the strongest denaturants used in physiochemical studies of protein folding. Guanidine Hydrochloride has the ability to decrease enzyme activity and increase the solubility of hydrophobic molecules. It is also a severe irritant so wear gloves when handling it. Brandon: looks kinda like urea hence protein unfolding I think

What's a guanylyl transferase?

Guanylyl transferases are enzymes that transfer a guanosine mono phosphate group, usually from GTP to another molecule, releasing pyrophosphate. Many eukaryotic guanylyl transferases are capping enzymes that catalyze the formation of the 5' cap in the co-transcriptional modification of messenger RNA.

Eukaryotic transcription - activating transcription factors and enhancers Eukaryotic DNA binding domains 4. Basic Helix-loop-helix/bHLH

Id is a family of proteins that heterodimerize with basic helix-loop-helix transcription factors to inhibit DNA binding of bHLH proteins. They contain the HLH dimerization domain but lack the basic DNA-binding domain and thus inhibit these proteins.

B-form DNA is an idealized structure. Tell me why this is the case

High resolution X-ray crystallographic structures of double-stranded DNA oligonucleotides of defined sequences show some variations in local structure, which are determined by the DNA sequence: - In particular the stacking of base-pair steps (i.e. two successive basepairs) varies in three parameters: twist, roll and slide - The dimensions of major and minor grooves also vary These sequence-dependent structural variations from the idealized B-form helix provide some basis for sequence-dependent DNA recognition and may aid initial docking of a protein onto DNA. However, true sequence specific recognition involves "probing" into the grooves of the DNA...

Genetically Modifying Tomatoes back then 1990s?

Modified tomatoes were on the market. Downregulated expression of the polygalacturonase gene (E.g. by expressing an antisense gene) - normally this degrades pectin, a component of the cell wall, causing the fruit soften. So downregulating gene expression delayed the ripening process.

Tell me about splicing

The coding sequences of many eukaryotic mRNAs are not continuous: mRNAs are transcribed as long precursors (pre-mRNAs) from which non-coding introns are removed during the process of splicing. The sequences left in the mature mRNA are called exons (note that not all exons code for proteins, as some of them contain the information for the 5' and 3' untranslated regions of mRNAs). Most mammalian genes contain introns, although some lack them (for example, genes encoding heat shock proteins and interferon). The number of exons ranges from 1 to 363 (with an average of 9). Exons tend to be smaller than introns (from 3 to 17,000 nucleotides, with an average of 150 for exons, compared to 70 to 800,000 nucleotides with an average of 3,000 for introns). Broad Topics: A) Factors needed for Splicing B) Spliceosome C) Alternative Splicing and Regulating Alternative Splicing D) Experiments for Splicing E) Factors that Regulate Splicing More Detailed Topics: 1) Discovery 2) Cis sequence elements for pre-mRNA splicing (in mammals) 3) Trans factors required for pre-mRNA splicing 4) Functional analysis of splicing cis elements and trans factors: 5) In vitro analysis of splicing reaction 6) The chemistry of splicing 7) The spliceosome 8) Regulation of splicing by RNA-binding proteins 9) The splicing code 10) Alternative splicing 11) Functions of alternative splicing 12) Functions of alternative splicing - example 1: the SV40 T antigen 13) Functions of alternative splicing - example 2: the Drosophila DSCAM gene 14) Functions of alternative splicing - example 3: the Drosophila sex-lethal RNA 15) Regulation of alternative splicing: 16) Regulation of alternative splicing - example 1: intron retention in S. cerevisiae 17) Regulation of alternative splicing - example 2: Transformer splicing by the SXL protein 18) Splicing and genetic disease

Prokaryotic Transcription Mechanism Transcription Basics?

Promoter specifies TSS: - consists of cis elements, usually shortly upstream of TSS - recognised by RNA Polymerase and/or associated factors

Tell me about EF-G

is a prokaryotic elongation factor involved in protein translation. As a GTPase, EF-G catalyzes the movement (translocation) of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome

Pleckstrin homology domain

is a protein domain of approximately 120 amino acids that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton This domain can bind phosphatidylinositol lipids within biological membranes (such as phosphatidylinositol (3,4,5)-trisphosphate and phosphatidylinositol (4,5)-bisphosphate),[8] and proteins such as the βγ-subunits of heterotrimeric G proteins,[9] and protein kinase C.[10] Through these interactions, PH domains play a role in recruiting proteins to different membranes, thus targeting them to appropriate cellular compartments or enabling them to interact with other components of the signal transduction pathways.

Tell me about Multiple cloning sites in general

is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. The purpose of a MCS in a plasmid is to allow a piece of DNA to be inserted into that region

Eukaryotic transcription - activating transcription factors and enhancers Give me an overview boy

The combination of RNA Pol II, general transcription factors and core promoter elements are not sufficient for transcriptional initiation in vivo, at least in part due to the repressive packaging by chromatin. Additional regulatory transcription factors are required, which bind at regulatory cis-elements and activate transcription in vivo by a variety of mechanisms. These regulatory transcription factors give rise to tissue-specific and developmentally regulated transcription. In this concluding section, we look at experimental methods for identifying necessary cis elements in vivo, the nature of these regulatory elements and the activator proteins that bind them, and finally consider the mechanisms by which they activate transcription in vivo.

Tell me about Sybr Gold

The migration of the DNA in the gel is detected by staining with Sybr Gold, a proprietary, asymmetric cyanine dye that exhibits >1000-fold fluorescence enhancement upon binding to nucleic acid - which is considerably (>x100) more sensitive than ethidium bromide (week 1).

What is a pronucleus?

is the nucleus of a sperm or an egg cell during the process of fertilization, after the sperm enters the ovum, but before the genetic material of the sperm and egg fuse.

What is IPTG?

iso-propyl-β-D-thio galactoside An inducer of lacZ, controls activity of PlacZ IPTG also controls Ptac, and T7 promoter IPTG inactivates lac repressor IPTG, unlike allolactose, is not hydrolyzable by β-galactosidase. Mechanism: Like allolactose, IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. But unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer.

Nucleic acid melting experiment. You measure the absorbance of DNA under different solvents, trying to measure its melting point under different solvents. How would you plot your graph?

The most useful way is to use the starting A260 as A0 and the A260 at temperature T as AT then plot AT/A0 against temperature.

What is Tris HCl?

Tris HCl is an organic compound used in buffer solutions. Picture shows Tris itself. tris(hydroxymethyl)aminomethane Tris contains a primary amine and thus undergoes the reactions associated with typical amines, e.g. condensations with aldehydes.

What is the melting temperature? (Tm) DNA

Under standard conditions the temperature at which half the DNA is melted is known as the melting temperature (Tm) You might consider why the Tm can be used as an estimate of the percentage GC in any given DNA sample (this can vary between 30% and 70% depending on the organism of origin).

What characteristics of E. coli are desirable in molecular cloning?

1) High Efficiency of Transformation a) ability to take up DNA b) lack of enzymes to degrade it once taken up (E.g. restriction enzymes) 2) Recombination deficiency to maintain plasmid stability 3) Debilitation (What is this??) BL: I think this is containment? 4) Other markers (may be part of 3))

What is the Basic Cloning Procedure?

4.1 The basic reaction 1) Take amplified PCR product 2) Have a linearised vector - E.g. pBluescript 3) Mix and Ligate - generates 3 main classes of molecule: 4) Transform E. coli with the ligation products 5) Plate on medium containing ampicillin, IPTG (iso-propyl-ß-D-thio galactoside - an inducer of lacZ) and X-gal. Then selective media will enable us to select plasmids with inserts...

Tell me 4 different ways you can clone a PCR product into a vector

5.1 Blunt cloning 5.2 'TA cloning' 5.3 Using restriction enzymes (pp2-8) 5.4 Gibson's assembly method

Some messages don't have poly(A) tails. Which? How could you make cDNA from them?

mRNAs from some eukaryotic genes (e.g. histones), chloroplast genes and bacterial genes generally lack polyA tails. If we know some of the sequence of the target mRNA we can use a primer specially synthesized for the purpose. If not, we can use primers of random sequence (often hexamers,containing all possible sequences). They will anneal all over the target mRNA at random.

Processing of pre-mRNA 3' end processing 3' end processing occurs cotranscriptionally. Tell me about it.

Although cleavage and polyadenylation can take place independently in vitro, they are closely linked to transcription in vivo. The evidence for this comes from experiments similar to those discussed for the capping enzymes: 1) Cells expressing ∆CTD forms of RNA Pol II are defective in processing 2) CPSF and CstF bind to CTD affinity columns

Other things to think about when expressing proteins in E. coli (pp173-178)

• Presence of introns - transcript processing § Won't get splicing etc. in E. coli. (Use cDNA?) • Translation - use of rare tRNAs? § May require high levels of rare tRNAs. Remember also that the universal genetic code isn't universal. • Fate of the protein after synthesis § May form inclusion bodies (easy to purify, but may not be easy to solubilize without loss of biological activity). Formation of inclusion bodies from improperly folded protein may depend partly on the redox state of the cytoplasm (can you think why?) so manipulation of that may help. § May get proteolysed. One of the major culprits in E. coli is the lon gene product, so use of lon- cells may help, but this can lead to other side effects, such as mucoidy and formation of long filamentous cells, which may be inviable. There is an outer membrane protease (product of ompT) that can also be a problem. § May get secreted. This may assist purification, and may help to avoid proteolysis. The MalE protein is secreted, so a fusion which includes the leader sequence of MalE should be exported to the periplasm. Other leader sequences include OmpA, OmpF, PhoA, β-lactamase (or PelB from Erwinia carotovora). § May need to be glycosylated for biological activity. • Effect on the host - toxicity § May kill it... this tends to cut down your yields

Fusion proteins can be useful for purification and other applications downstream:

• Protein purification - binds a specific column or matrix (E.g. Glutathione-S-transferase (GST), His-tag, maltose binding protein (MBP) etc.) • Protein solubility - stops aggregation (E.g. MBP) • Tagging proteins - E.g. GFP tag, 'epitope tagging' (E.g. Flag, Myc, HA etc.) - useful for various applications (E.g. Immunoprecipitations, immunofluorescence, immunoblots etc.) - see later. • 'Pull down' assays - can immobilize a recombinant fusion protein (E.g. GST or His-tagged) on a column then look for interactions with other proteins - see later. • Phage display - produce a fusion with a phage coat protein - displayed on the surface - useful for identifying interacting ligands etc. If your vector is designed to produce a fusion protein with the extra bit at the N-terminus, it typically contains appropriate translation initiation signals etc.

What is a degenerate primer?

A mix of oligonucleotide sequences in which some positions contain a number of possible bases, giving a population of primers with similar sequences that cover all possible nucleotide combinations for a given protein sequence

Tell me about ampicillin's mechanism of action

Ampicillin is in the penicillin group of beta-lactam antibiotics and is part of the aminopenicillin family. Ampicillin is able to penetrate Gram-positive and some Gram-negative bacteria. That amino group, present on both ampicillin and amoxicillin, helps them enter the pores of the outer membrane of Gram-negative bacteria, such as E. coli, Proteus mirabilis, Salmonella enterica, and Shigella. Ampicillin acts as an irreversible inhibitor of the enzyme transpeptidase, which is needed by bacteria to make the cell wall. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell lysis; therefore, ampicillin is usually bacteriolytic

What's the deal with ddATP?

BL: I guess it can be hydrolysed but not elongated? It is used in experiments involving the poly(A) tail

The major groove is information-rich - Why?

BL: I think coz proteins can work out what the bases are from the major and minor grooves, because the "feel" of the bases are kinda exposed here, so proteins can "feel" what the bases are at the major and minor grooves Even though the formation of W-C base-pairs uses complementary H-bonding groups that distinguish the four bases, and the four base-pairs are isomorphic (same shape), there is still "spare" chemical information that can be used for sequence-specific recognition of base-pairs. Note the distribution of available hydrogen bond donors (D) & acceptors (A), Me groups (M) and non-polar hydrogens (H) on the 4 different base-pairs On the major groove side, these allow discrimination of all 4 base-pairs (MADA vs ADAM vs DAA vs AAD) i.e. sequence-specific recognition In contrast, the minor groove is information poor: (AHA vs AHA vs ADA vs ADA). For instance, comparing T=A vs A=T base-pairs, in both cases the minor groove is flanked by H bond acceptor groups — O2 of T and N3 of A. Only the position of one non-polar hydrogen (i.e. not available for H-bonding) varies slightly. Moreover, the major groove is wide, shallow and accessible to amino acid sidechains Consequence: in principle, sequence specific recognition could be via the major groove (see slides 68, 69 for a specific example with the CAP protein). NB there are some structural polymorphisms in groove dimensions of B-DNA. e.g. Minor groove narrower in A/T rich sequence, wider in G/C rich sequence

Translation in eukaryotes

Because elongation and termination are very similar in prokaryotes and eukaryotes, we will concentrate on the process of eukaryotic initiation Similarities and differences between eukaryotic and prokaryotic initiation How do we study translation? The reticulocyte in vitro system Initiation in eukaryotes: the scanning model Initiation in eukaryotes: mechanism and factors Role of the poly(A) tail in translation Scanning

Absorption of nucleic acids is at 260 nm.... but what other factors affect it?

Both hydrogen bonding and base-stacking interactions decrease this absorption by the bases. So the absorbance of intact, native, nucleic acid molecules is less than that of the nucleotides of which they are composed. On disrupting the secondary structure of DNA either by heating or by exposing it to strongly alkaline pH, the absorption of light at 260 nm (A260) rises to approximately 1.4 times its original value. This is known as the hyperchromic effect. The extra hydroxyl group on the 2' position in the backbone of RNA makes it sensitive to alkali hydrolysis, so changes in RNA structure are induced by heating. Brandon: when decomposed, the nucleotides take up more space in solution. There is also a larger SA:vol ratio, so perhaps this accounts for the greater absorbance seen in decomposed nucleotides.

Why is DNA more likely to be incompletely denatured with excess magnesium?

Brandon hypothesis: neutralise DNA negative strands, so now DNA is neutral, so won't repel opposite strand as powerfully, so more likely for double strand dna to remain

T4 DNA ligase

Brandon: T4 infects E coli and thats probably why you use T4 DNA ligase. T4 ligase is the ligase most-commonly used in laboratory research. It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. Unlike E. coli DNA ligase, T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor.

Purifying DNA after PCR and restriction enzyme digests

After the PCR you will purify the amplified DNA fragment to remove enzymes and primers up to 40 nts long and dNTPS. For this you used a QIAquick spin column®. This is a silica membrane assembly that efficiently binds single or double stranded DNA of more than 100 bp in a high salt buffer that contains a high concentration of chaotropic salts. Chaotropic agents disrupt the hydrogen-bonding network of water, destabilizing the DNAwater hydrogen bonds. The DNA can then establish binding interactions with the silica surface. Washing with a high percentage of ethanol removes unbound impurities, while enhancing the DNA binding. The DNA is then eluted with a low-salt buffer. The same procedure is followed after the restriction enzyme digest, in this case to remove the small fragments that the restriction enzymes have removed from the PCR products as well as the enzymes themselves. Brandon: silica membrane is negatively charged. With right buffer you can probably change the charges so silica and DNA DO bind, however changing the buffers again you can get both the DNA and silica to be negatively charged so DNA can be removed. I think thats how it works.

Tell me about allolactose

Allolactose is a disaccharide similar to lactose. It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose. It is an inducer of the lac operon in Escherichia coli and many other enteric bacteria. It binds to a subunit of the tetrameric lac repressor, which results in conformational changes and reduces the binding affinity of the lac repressor to the lac operator, thereby dissociating it from the lac operator. The absence of the repressor allows the transcription of the lac operon to proceed. A non-hydrolyzable analog of allolactose, isopropyl β-D-1-thiogalactopyranoside (IPTG), is normally used in molecular biology to induce the lac operon.

Tell me about CLIP

combines UV cross-linking with immunoprecipitation in order to analyse protein interactions with RNA or to precisely locate RNA modifications (e.g. m6A). CLIP-based techniques can be used to map RNA binding protein binding sites or RNA modification sites of interest on a genome-wide scale, thereby increasing the understanding of post-transcriptional regulatory networks. HITS-CLIP, also known as CLIP-Seq, combines UV cross-linking and immunoprecipitation with high-throughput sequencing to identify binding sites of RNA-binding proteins. CLIP-seq depends on cross-linking induced mutation sites (CIMS) to localized protein-RNA binding sites. Because CIMS are reproducible, high sequencing depths allow CIMS to be differentiated from technical errors.

Tell me about vapour diffusion crystallisation

common method of protein crystallisation Droplet containing purified protein, buffer, and precipitant are allowed to equilibrate with a larger reservoir containing similar buffers and precipitants in higher concentrations. Initially, the droplet of protein solution contains comparatively low precipitant and protein concentrations, but as the drop and reservoir equilibrate, the precipitant and protein concentrations increase in the drop. If the appropriate crystallization solutions are used for a given protein, crystal growth will occur in the drop This method is used because it allows for gentle and gradual changes in concentration of protein and precipitant concentration, which aid in the growth of large and well-ordered crystals. require sealing of the environment so that equilibration between the drop and reservoir can occur

Chromatin Packaging DNase I sensitivity reflects gene expression

Blurry: Digest chromatin with DNase I Extract DNA and cleave with restriction enzyme Electrophorese fragments and denature DNA; probe for expressed and nonexpressed genes Probe 1 Probe 2 Compare intensities of bands in preparations in which chromatin was digested with increasing concentrations of DNase Dnase Probe 1 DNA is preferentially digested Probe 2 DNA is not preferentially digested. Extra: Sensitivity of DNA in chromatin to digestion by DNase I was an early form of biochemical evidence indicating a change in chromatin packaging in expressed genes compared to non-expressed genes. The first experiment showed that expressed genes are digested by DNase I about ~5-fold more rapidly than bulk chromatin. This extends across the length of the gene. However, they are still occupied by nucleosomes (see e.g. slide 80), so the enhanced DNase sensitivity may reflect chromatin de-condensation, perhaps by a 30 nm to 10 nm fibre transition (see following slides) The DNase I hypersensitivity assay provides a higher resolution view showing specific locations that are digested by DNase I, 10-20 fold faster than the rest of the gene. These really are nucleosome-free regions, due to binding of transcription factors at promoters or enhancers. The genes are sometimes "poised" to be activated rather than continually active. The poised state, with DNase hypersensitive sites could be established at the previous round of DNA replication. NB: nucleosome-free regions were not seen in slide 80, because the plot shows the average density of MNase resistant nucleosome tags across thousands of genes. The position of DNase hypersensitive, nucleosome-free, sites varies between genes & is not at a fixed location with respect to the transcription start site.

What is lysogeny?

Lysogeny, or the lysogenic cycle, is one of two cycles of viral reproduction (the lytic cycle being the other). Lysogeny is characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome or formations of a circular replicon in the bacterial cytoplasm. In this condition the bacterium continues to live and reproduce normally. The genetic material of the bacteriophage, called a prophage, can be transmitted to daughter cells at each subsequent cell division, and at later events (such as UV radiation or the presence of certain chemicals) can release it, causing proliferation of new phages via the lytic cycle.

Hyaluronic Acid

Made of glucoronic acid and n-acetyl glucosamine distributed widely throughout connective, epithelial, and neural tissues.

DAPI

a fluorescent stain that binds strongly to adenine-thymine rich regions in DNA. It is used extensively in fluorescence microscopy. As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore the effectiveness of the stain is lower. Brandon: Why does it bind to AT rich regions.... probably electrostatic attractions similar to base stacking...

What is heparin?

a highly-sulphated glycosaminoglycan.

What is a non-sense mutation?

a point-nonsense mutation is a point mutation in a sequence of DNA that results in a premature stop codon

Tell me about the C2 domain

a protein structural domain involved in targeting proteins to cell membranes. The typical version (PKC-C2) has a beta-sandwich composed of 8 β-strands that co-ordinates two or three calcium ions, which bind in a cavity formed by the first and final loops of the domain, on the membrane binding face. Many other C2 domain families don't have calcium binding activity The picture is an example of a β protein

mineralocorticoids

Mineralocorticoids are a class of corticosteroids, which in turn are a class of steroid hormones. Mineralocorticoids are produced in the adrenal cortex and influence salt and water balances (electrolyte balance and fluid balance). The primary mineralocorticoid is aldosterone

Eukaryotic transcription - core promoter and general transcription factors (GTFs) Roles of General Transcription Factors TFIIF, TFIIH, Mediator

Note that the GTFs play a functionally equivalent role to bacterial σ factors in vitro. Pol II alone cannot accurately initiate transcription, despite having 12 subunits. Addition of GTFs (25 protein subunits) confers accurate initiation. Note that some of the properties of σ subunits are spread across different GTFs - TBP specifically recognizes core TATA elements - TFIIF represses non-specific DNA binding RNA Polymerase II is also unique (compared to prokaryotic RNAPs and RNA Pol I and III) in requiring GTFs even after it has been recruited to the promoter: TFIIE and TFIIH. While the GTFs formally confer σ like properties in vitro, in intact cells RNA Pol II, GTFs and core promoter elements are not sufficient....

What sequence does BamHI recognise and cut?

Note that these recognition sites are palindromic, so that the resulting overhanging ends are identical when read from 5' to 3'. Also notice that these recognition sites contain 6 specific bases, so they are expected to occur once in every 4⁶ base pairs or roughly 4kbp

What sequence does HindIII recognise and cut?

Note that these recognition sites are palindromic, so that the resulting overhanging ends are identical when read from 5' to 3'. Also notice that these recognition sites contain 6 specific bases, so they are expected to occur once in every 4⁶ base pairs or roughly 4kbp

You did some vector cloning. And you are doing some blue white screening on a plate. How would you assess the efficiency of ligation?

Now calculate the percentage of blue and white colonies on each plate.

Prokaryotic Transcription Mechanism Domains of σ subunit

σ alone does not bind because 1.1 stops binding when not in a holoenzyme

X-ray crystallography: But in order to calculate the electron density by Fourier synthesis, we need to derive the relative phase of each reflection: This can is usually derived by three principal methods:

- Introduction of heavy metal that changes the phase - Using the ability of certain elements to absorb X-rays and change the diffraction intensities. Seleno-Methionine can be incorporated in the place of methionine during protein expression (typically in E.coli). - Using a (closely) related protein structure to derive calculated phases

Tell me about DNA structure - base pairing

- Double-stranded right-handed double-helix - Backbone - alternating sugar (deoxyribose) and phosphate groups linked by phosphodiester bonds to 5' and 3' groups of adjacent sugars - Antiparallel chains (5' to 3' and 3' to 5') - Bases joined by N-glycosidic bond from N1 of Py or N9 of Pu to ribose C1' - Bases primarily exist as the amino and keto tautomers (vs. imino & enol) - Watson-Crick base-pairs G=C, C=G, A=T, T=A specified by complementary H-bonding - The 4 base-pairs are isomorphic - consistent with uniform DNA structure - Base-pairing by H-bonding provides stability and specificity

Histone handshake motif?

Core histone fold domain contains 3 alpha helices, and these have extensive hydrophobic interactions allowing the histones to bind to each other forming heterodimers (handshake motif) and form a histone octamer.

Beta lactam ring

Core structure of penicillins (top) and cephalosporins (bottom). β-lactam ring in red.

Genetic engineering lawyer defintion?

"the formation of new combinations of heritable material by the insertion of nucleic acid molecules, produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur, but in which they are capable of continued propagation". Brandon: Formation of new DNA combinations using vectors, in hosts where this DNA does not naturally occur.

3.1 So what does a typical cloning vector look like? (pp52-57, 66) An example of a standard lab "workhorse" vector is pBluescript: pBluescript has a number of features common to other cloning vectors, as follows:

(i) The Multiple Cloning Site (MCS) (ii) The lacZ' gene (iii) Antibiotic resistance - E.g. ampicillin (Has the bla gene)

Prokaryotic Transcription Regulation Negative regulation by Lac repressor

- Lac repressor: tetramer of 38 kDa subunits - dimer of dimers (slide 58) - Operator recognised by N-terminal helix-turn-helix motifs of one dimer docking into major groove (slide 58) - Blocks RNAP binding (some in vitro evidence suggests blocks promoter melting) - Additional repressor binding sites at +410 and -80. At least one required for full repression i.e. tetramer binds two palindromic sites, looping out intervening DNA - Inducers such as allolactose (Gal-β(1-6)-Glc) bind repressor & reduce affinity for operator (allolactose produced by β-Galactosidase itself) - Natural inducers metabolized by β-Gal - activation only persists while lac available - Artificial "gratuitous" inducers bind repressor but not metabolized by β-Galactosidase e.g. IsoPropylThioGalactoside (IPTG) - Repressor only active at Lac operon - Only ~10 repressor tetramers/cell

DNA structure Tell me about Stacking and Electrostatic Repulsion

- Stacking of adjacent base-pairs (π electron rings) provides stability to double-stranded structure - entropically favoured exclusion of water - Stacking maximized by "propellor twist" of 16-18⁰ Bases within base-pair do not lie precisely on same plane - Stacking favours extended DNA conformation - bending reduces stacking - Stacking is optimal in 5' Purine-Pyrimidine 3' bp steps: Maximal stacking in 5'-GC-3' Minimal stacking in 5'-TA-3' - Sequences such as TATA readily melted - fewer H-bonds & lower stacking - Electrostatic repulsion between negatively charged backbone phosphates: i) inter-strand: destabilizes double helix - reduced by higher ionic strength ii) intra-strand: favours an extended conformation - can be countered by bound proteins, which can facilitate bending

One Gene at a time or Genome-wide analyses

- post-genomic era: global analyses (e.g. microarrays, deep sequencing) often provide general insights not readily obtainable by older "one gene at a time" methods - new hypotheses can be generated via computational - "in silico" analyses of genome- transcriptome- or proteome-wide data-sets - hypotheses subsequently tested in representative model system by traditional in vivo and in vitro approaches

Modification Interference: Missing bands indicate where protein binds. Whats the experimental reason why there are no bands here?

1 strand labelled at only 1 end. DNA chemically modified, but only 1 chemical modification per DNA strand. then you incubate with the protein. If modification is at protein binding site, protein won't bind. So these DNA molecules have no protein attached to them. Other DNA strands will have 1 modification AND protein binding at same time because modification is somewhere else on the DNA. DNA that is bound by protein are separated from DNA that has no protein bound (by gel shift). This means you only isolate the DNA that was bound by protein. This means when you remove the proteins from the DNA, and cleave the DNA, and run the result on a gel, missing bands arise because you couldn't get a DNA molecule of that specific length because you don't have any DNA with modifications (and therefore cleavage points) at that site, so you don't get any bands of that length. This is because you removed the DNA strands that could never be bound by DNA in the first place.

Translation in eukaryotes Similarities and differences between eukaryotic and prokaryotic initiation

1) Both use a start codon and a dedicated initiator tRNA 2) Both bind the small subunit to the RNA first, with the assistance of initiation factors 3) The recognition of the mRNA and the initiation codon is completely different: a) In prokaryotic cells the small subunit binds at the SD box and the initiation codon b) Eukaryotic 18S rRNA is similar to bacterial 16s rRNA but does not have the sequence that pairs with the SD box c) In eukaryotes the small subunit binds the cap and scans the mRNA until the first initiation codon 4) Both GTP and ATP are required in eukaryotes (only GTP in prokaryotes)

Regulation of translational efficiency in bacteria Regulation of gene expression in bacteria by small non-coding RNAs (sRNAs)

1) 50-200 nucleotides 2) ~100 different species in E. coli 3) Can have a single target or many 4) Bind to mRNAs with complementary sequences and regulate their translation or stability 5) Usually work in trans 6) Usually produced as full transcripts (i.e., not processed from precursors - see miRNAs later) 7) Often function in a complex with an RNA-binding protein called Hfq sRNAs can activate translation by competing with and disrupting secondary structures that limit accessibility to the SD sequence. They can also repress translation by directly binding and masking the ribosome-binding site

SAQ List the steps involved in the generation of 'knockout' mice.

1) Add lox sites flanking gene to be knocked out 2) Use Cre recombinase

Regulation of translation in eukaryotic cells Control of the eIF4E-eIF4G interaction

1) As discussed above, eIF4E binds to the cap and recruits eIF4G 2) This interaction can be modulated by 4E-BPs (4Ebinding proteins), which compete with eIF4G for the binding to eIF4E, thus inhibiting initiation 3) The binding affinities of 4E-BPs to 4E are regulated by phosphorylation. For example, phosphorylation of 4E-BP by Tor kinase releases 4E, promoting translation and cellular growth 4) 4E-BPs can also regulate translation in a transcriptspecific way (see below)

What are the three types of interactions that determine the stability of a double stranded DNA molecule? How does each of the interactions depend upon the sequence of bases in the DNA?

1) Base Stacking - stacking of adjacent base pairs (pi electron rings) provides stability to double stranded structure, entropically favoured exclusion of water. Stacking maximized by propeller twist of 16-18 degrees. There is maximal stacking if a purine is followed by a pyrimidine, and this repeats. However, GC base stacking is stronger than TA base stacking. 2) Hydrogen Bonding. More GC pairs, the stronger H bonding is. 3) Electrostatic repulsion between negatively charged phosphate backbone. This destabilises the double helix inter-strand, whereas intra-strand, it favours an extended conformation of the DNA. Sequence doesn't really affect electrostatic repulsion. TBC!!!!!!!!!! What does therese say in her supervision?

Eukaryotic small regulatory RNAs Regulation of gene expression by miRNA and siRNAs

1) Both miRNAs and siRNAs function as part of the RISC complex 2) The miRNAs impart specificity to the complex by forming base pairs with their target mRNAs 3) In general, for siRNAs and plant miRNAs complementarity with their targets is perfect and matching sequences are long; this leads to endonucleolytic cleavage and decay of the target mRNA 4) In animals the matching sequences tend to be short (limited to a region of the miRNA called the seed) and complementarity is usually not perfect. a) Their targets are translationally repressed (probably at the level of initiation, although there are other models) and can also be degraded (although not by endonucleolytic cleavage) b) Some recent experiments suggest that degradation may be prevalent

Eukaryotic transcription - core promoter and general transcription factors (GTFs) The RPB1 C-Terminal Domain (CTD)

1) CTD is flexible - easily proteolytically cleaved, not seen in X-ray structure 2) 2 main forms of RPB1 distinguished on SDS-PAGE: a) Pol IIa - CTD hypo-phosphorylated - associated with transcription initiation b) Pol IIo - CTD hyper-phosphorylated - associated with elongation (Also smaller Pol IIb, in which CTD has been proteolytically cleaved) 3) Major phospho-acceptors are Ser-2 and Ser-5 (YSPTSPS) 4) CTD essential for viability 5) CTD binds initiation, elongation factors, histone modifying enzymes and RNA processing factors (capping, splicing, 3' end processing) 6) CTD joined to body of Pol II close to RNA exit channel

Processing of pre-mRNA 5' end processing: capping Tell me about Cap structure and its 4 functions

1) Cap structure: N7-methyl-guanosine, attached to the 5' end of the mRNA through a 5'-5' triphosphate bond 2) The cap is not encoded in the DNA 3) Functions: a) Increases the efficiency of splicing of 5' proximal introns b) Required for export to the cytoplasm c) Necessary for efficient translation initiation d) Protects mRNA from 5' exonucleases

Genome-wide approaches to study gene expression A genome-wide view of splicing

1) Case study: The Nova proteins are a family of brain-specific RNA-binding proteins that regulate alternative splicing. They can enhance or repress inclusion of an exon 2) A comprehensive genome-wide view of the function of Nova requires the systematic identification of its target RNAs (and where on these targets it binds) as well as the effects of the binding on target alternative splicing 3) Identification of the targets and binding sites of RBPs: CLIP-seq (Cross-Linking and ImmunoPrecipitation - deep sequencing) a) Proteins and RNA are cross-linked in vivo using UV light b) An extract is prepared from the cross-linked tissue c) The extract is treated with RNase; only the RNA fragments protected by protein (which are the protein binding sites) survive the treatment d) Nova (orange in the figure) is purified using specific antibodies together with associated RNA fragments (its binding sites) e) Nova protein is removed, the RNA is purified and analyzed by deep sequencing f) The sequence reads are mapped to the genome g) This results in the identification of all binding sites of the nova protein (the resolution is very high, so the binding sites of the protein can be mapped with respect to the consensus sequences of each exon and intron) 4) The location of the binding does not provide information on the functional consequences of the binding event (that is, if it promotes or represses splicing, or if it has no functional significance). Thus, it needs to be complemented with functional studies. One powerful approach is the use of splicingsensitive DNA microarrays: a) Splicing-sensitive microarrays contain probes for exon-exon and exon-intron junctions. Therefore, they can be used to quantify the frequency of each alternative splicing event for the whole genome b) The microarrays are used to compare splicing between normal cells and cells in which Nova (or any other protein) has been inactivated: this can be used to produce a map of all splicing events functionally regulated by Nova (including whether it activates or represses splicing) 5)Integration of the data: the binding and functional data are integrated to produce functional maps. The figure below shows the binding pattern for an exon whose splicing is promoted (top, in red) or repressed (bottom, in blue) 6) Conclusion: Whether Nova activates or represses splicing depends on its position. This is a general principle, true for other RBPs For clearer picture go mata page 44 Picture interpretation: plate that binds to particular sequences in introns or exons. If exon-intron-exon binds it gives a different colour to if exon-exon binds. depending on the colour from each tile of the plate, it will tell you if both exon-intron-exon or exon-exon binds. Maybe I haven't fully deciphered this picture yet.

Processing of pre-mRNA 3' end processing Tell me about Formation of the poly(A) tail

1) Cis sequences involved: a) AAUAAA ~12-30 nucleotides upstream of cleavage site, highly conserved b) U-rich or GU-rich sequences up to 30 nucleotides downstream of the cleavage site (Downstream Sequence Element or DSE) 2) 3' end processing can be studied in vitro: The assay requires a radiolabelled RNA substrate, nuclear extracts and ATP. The assay can be used to show that cleavage and polyadenylation can occur independently (see below). 1] In the presence of ATP, the substrate is cleaved and polyadenylated. 2] In the presence of ddATP, the substrate is cleaved but not polyadenylated. 3] An RNA molecule that mimics a cleaved substrate is polyadenylated but not cleaved 3) The in vitro system can also be used to show that polyadenylation occurs in two stages. The first stage requires requires AAUAAA sequence and leads to the addition of ~10 adenines. The second stage is independent of AAUAAA but requires the A tail already present, and leads to the addition of a long poly(A) tail. Evidence: a 'cleaved' substrate is polyadenylated, but not if the AAUAAA sequence is mutated. However, a substrate with a mutated AAUAAA and a short poly(A) tail is adenylated further 4) Trans factors controlling 3' end processing Processing of the 3' end is carried out by a very complex machinery. Purification and mass spectrometry analysis of the human 3′ end processing complex revealed the presence of ~85 polypeptides. Of these, ~50 are involved in coupling 3'-end processing to other processes. The main factors are the following: a) CPSF (Cleavage Polyadenylation Specificity factor): made up of 5 subunits (CPSF73 is the endonuclease). It binds to binds to AAUAAA and CStF and is required for both cleavage and polyadenylation b) CStF (Cleavage Stimulation Factor): it binds the GU/U repeats and is only required for cleavage. CPSF and CStF interact with each other and bind to the pre-mRNA cooperatively c) Poly(A) polymerase: Adds adenylates to 3' end of the mRNA and is also required for cleavage

Investigating Mechanisms of Gene Expression

1) Clone & sequence the gene (genomic or cDNA): see lectures by Deirdre Scadden 2) Develop assay system - in vivo or in vitro 3) Identify cis-acting sequences - essential DNA or RNA sequences that typically act as binding sites for trans-acting protein (or RNA) •consensus sequences • mutations 4) Identify trans-acting factors (protein or RNA) that bind cis-sequences 6) How do cis elements and trans factors combine to control function? NB: cis and trans are used here in the Genetics rather than the Chemistry sense

Translation elongation in prokaryotes Binding of the aminoacyl-tRNA to the A site

1) EF-Tu is a monomeric GTP-binding protein with GTPase activity. It binds GTP and an aa-tRNA to form a ternary complex analogous to that formed by IF2. The binding of EFTu masks the aminoacyl group in the aa-tRNA, preventing it from reacting with the peptidyl-tRNA 2) The ternary complex joins the ribosome at the A site. If the codon-anticodon match is incorrect, the ternary complex is released without GTP hydrolysis. If the match is correct, a conformational change in the ribosome triggers GTP hydrolysis and the release of EF-Tu•GDP. The aminoacyl end of the tRNA in the A site moves towards the peptidyltransferase centre (this process is called accommodation)

Eukaryotic small regulatory RNAs Biogenesis of siRNAs:

1) Formed from double stranded RNA precursors 2) Precursors are generated by viral infection, transposons, sense-antisense transcript pairs 3) They are also formed when exogenous dsRNA is introduced in a cell (RNAi) 4) They are cleaved by Dicer in the cytoplasm (Drosha is not involved) and loaded onto the RISC complex

4 examples of fusion proteins used for protein purification

1) Fusion with a 6x His-Tag 2) Fusion with Glutathione S-transferase Use of fusion proteins for increased solubility and purification: 1) Fusion with MalE maltose binding protein (MBP) Epitope tag is also a fusion protein?

Translation elongation in prokaryotes Formation of the peptide bond

1) GTP hydrolysis and the release of EF-Tu•GDP bring together the aminoacyl and peptidyl ends 2) The formation of the peptide bond is catalysed by the ribosomal peptidyl-transferase activity, which is located on the 50S subunit 3) The enzymatic activity is performed by the rRNA (see later)

Splicing Tell me about the Functions of alternative splicing

1) Generation of variability ion protein coding information: Some genes can generate hundreds of distinct protein isoforms via alternative splicing. 2) Regulation of gene expression

Splicing Regulation of splicing by RNA-binding proteins

1) In addition to the consensus sites described above, splicing is controlled by regulatory sites called enhancers and silencers 2) Regulatory elements are classified according to their activity and location: a) Exonic splicing enhancers (ESEs) b) Intronic splicing enhancers (ISEs) c) Exonic splicing silencers (ESSs) d) Intronic splicing silencers (ISSs) 3) Regulatory elements have the following features: a) Gene-specific b) Recognised by regulatory proteins that modulate the interactions between nearby splicing sites and the spliceosome c) Can regulate constitutive or alternative splicing 4) Splicing enhancers and repressors form a 'splicing code' that regulates the efficiency of splice site usage 5) The code is 'read' by sequence-specific RNA-binding proteins that bind to cis-acting elements and modulate the recruitment of the spliceosome 6) Splicing Activators (another card) 7) Splicing repressors (another card)

Initiation of translation in prokaryotes. In vitro evidence for base pairing of SD sequence and 16S rRNA?

1) In vitro: a) A 37-nucleotide RNA fragment containing the initiation codon is labeled with ³²P and incubated with ribosomes, f-Met-tRNA and initiation factors under conditions that lead to initiation but not elongation b) The sample is treated with SDS, which denatures proteins and dissociates RNA-protein interactions. However, it does not affect RNA-RNA base pairing c) The samples are then separated by non-denaturing gel electrophoresis Result: The labeled RNA fragment co-migrates with 16S rRNA at ~1,600 nucleotides when it is preincubated with ribosomes, indicating that the two RNAs are bound by base pair interactions Note that this initiation mechanism allows the recognition of multiple ribosome binding sites on a single mRNA, and thus the use of polycistronic mRNAs.

Translation The translation Cycle

1) Initiation: Involves the recognition of the initiation codon and all other events up to the formation of the first peptide bond. This is a slow step, and usually rate-limiting. It is also the most frequent point of regulation 2) Elongation: Formation of all peptide bonds until the end of the coding sequence 3) Termination: Release of the polypeptide, dissociation of the ribosome from the mRNA

Splicing Regulation of alternative splicing - example 1: intron retention in S. cerevisiae

1) Intron retention is common in yeast, where more complex forms or alternative splicing are not present. Intron retention is generally used to switch off gene expression, avoiding the production of a protein from the unspliced transcript 2)The MER3 gene is transcribed in early meiotic cells, but is spliced very inefficiently and no protein is produced 3) In later stages of meiosis MER3 pre-mRNA is spliced, and MER3 protein is synthesized 4) How is this regulated? a) The 5' splice site in MER3 pre-mRNA is very weak, thus U1 snRNP binds very inefficiently b) The intron contains an intronic splicing enhancer (ISE) c) The RBP MER1 binds to the ISH and facilitates the recruitment of U1 snRNP d) MER1 is expressed only in meiotic cells, making the splicing meiotic-specific e) MER1 regulates the expression of a total of four meiotic RNAs in the same way 5) Evidence a)MER1 is required for efficient splicing: there is no splicing in MER1∆ cells b) Mutations in ISE abolish splicing c) Mutations that restore consensus at 5' SS lead to constitutive splicing (that is, MER1independent)

Splicing Splicing and genetic disease

1) Many inherited conditions are caused by mutations that affect splicing. These mutations can affect splicing in two ways: a) By inactivating 5' and 3' splice sites, enhancers or repressors b) By the generation of new splice sites 2) Some splicing regulatory sequences are located in exons, explaining why 'silent' mutations in coding regions can have phenotypic effects 3) Example: Hutchinson-Gilford Progeria Syndrome (HGPS) a) Very severe condition that causes premature aging and early death b) Caused by a point mutation in exon 11 the prelamin A gene (the precursor of lamin) c) The mutation activates a cryptic splice site d) The resulting protein has a 50-aminoacid deletion that removes a protease cleavage site. The mutant protein is not processed correctly and acts as a dominant negative e) Note that this syndrome is caused by a silent mutations (GGC->GGT = Glycine->Glycine)

Splicing Tell me about the splicing code

1) Most introns contain multiple regulatory sequences (enhancers and repressors) in addition to the core splice sites 2) What determines if a splice site is used? a) 'Strength' of splice sites (good splice sites are more similar to consensus) b) Presence/absence of enhancers and repressor elements and the proteins bound to them c) RNA secondary structure, which can mask splice sites 3) Note that the same protein can be function as activator or repressor depending on its location (see later) 4) An exon that is always included in the mature mRNA is called 'constitutive', while an exon that is not included in the final mRNA is said to be 'skipped'

Splicing Functional analysis of splicing cis elements and trans factors:

1) Mutations in the more highly conserved positions of consensus sequences either completely inactivate splicing or sometimes result in the use of nearby 'cryptic splice sites' that have similar sequences 2) Mutations in the introns and exons outside of consensus sequences usually have no effect 3) Removal of first eight nucleotides of U1 snRNA (that pair with the 5' SS) blocks splicing 4) The effects of some mutations in 5' splice site and branch point can be reversed by complementary mutations in U1 snRNA and U2 snRNA respectively (not in the 100% conserved GU of the 5'SS or branch point A). This provides direct evidence for base-pairing 5) The only other requirement is minimal distance between the splice sites (~70 nt) in order to allow productive binding of splicing factors 6) Antibodies against U1 snRNP block splicing 7) Removal of U snRNPs from nuclear extracts (see below) inactivate their ability to splice premRNA

RNA recognition

1) Non base-paired regions: all functional groups available for recognition by protein or RNA Sequence-specific recognition easy 2) Structurally diverse RNAs readily discriminated by shape 3) W-C base-paired dsRNA has A-form helix - information rich major groove inaccessible 4) Ends (2-3 bp from end) and distortions in helix open up major groove allowing sequence specific recognition 5) Long fully double-stranded RNA usually recognized as "foreign" e.g. viral

Advantages of PCR

1) PCR can be very specific, depending on the primers used. Brandon: I guess with a pool of DNA, only the DNA that has an incorporated primer will be amplified, hence its specific. 2) PCR can be readily automated 3) PCR can make use of DNA that may be partially degraded 4) PCR can work with really tiny quantities of DNA.

Difficulties with PCR? Name 3

1) Polymerase errors. Lack of 3'-5' exonuclease proofreading means Taq polyermase has a significant error rate (1 in 10⁴ nucleotides). Note that if an error occurs in an early cycle, it will be represented in a lot of the product molecules. Using DNA polymerase with 3' to 5' proofreading activtiy also has drawbacks... typically cost and yield. 2) Size of the product can be a difficulty. Brandon: 1 and 2 are linked. When wrong base introduced, Taq polymerase sometimes dissociates, limiting size of product. 3) non-specific priming: a common problem caused by primers annealing in the wrong place.

Eukaryotic transcription - activating transcription factors and enhancers Cis elements identified in vivo + General properties of upstream elements?

1) Promoter 3' end deletions: Delete TATA box: Efficiency modestly reduced, several start sites. Further deletions reduce efficiency 2) Promoter 5' end deletions: Eliminate transcription well before TATA box deleted - Core elements (& associated GTFs) are insufficient in vivo Deletions identify "Upstream Elements" (UE's) a) UE's precisely delineated by point mutations & internal deletions b) UE's necessary for transcription in vivo c) Binding sites for activating transcription factors

Eukaryotic transcription - core promoter and general transcription factors (GTFs) RNA Pol II Promoters

1) Promoter contains sequences necessary to specify transcription start sites (TSS) 2) TSS mapped by aligning cDNA sequences to genomic DNA

Translation in eukaryotes Scanning Mechanism?

1) Scanning involves two separate processes: ribosome translocation and unwinding of secondary structure 2) The 43S complex scans the mRNA until it reaches the first AUG 3) The helicase activity of eIF4A unwinds initial secondary structure. After that, the unwinding is done by eIF4A/B 4) The helicase activity requires ATP; this explains requirement of ATP for scanning (in vitro, ATP is only needed in the presence of secondary structure - i.e. translocation per se does not require ATP) 5) Once the AUG is recognised, GTP in eIF2-GTP is hydrolysed and released (together with other eIFs) 6) Scanning explains why eukaryotic mRNAs are monocistronic. Ribosomes scan from 5' cap and detach at termination, thus they cannot access downstream cistrons clearer picture on mata page 37

RNA secondary Structure

1) Secondary structure formation is primarily driven by W-C base pairing 2) X-ray crystallographic and cryoEM structures of ribosomes, tRNA, riboswitches, aptamers reveal numerous other structural motifs involved in tertiary folds 3) Structured RNA can be catalytic e.g. ribosome, spliceosome 4) Structured RNA can bind small molecule ligands - "riboswitches" 5) Mg2+ and/or proteins often needed for tertiary fold and catalytic activity of ribozymes

Regulation of translational efficiency in bacteria Regulation of translation by thermosensors

1) Secondary structures that block access to the SD sequence 2) The secondary structure is sensitive to changes in temperature 3) You will discuss an example of this type of regulator during your journal club

Eukaryotic small regulatory RNAs Introduction

1) Small RNAs (20-30 nucleotides) 2) They work together with a protein of the argonaute (ago) family 3) They regulate gene expression by forming base pairs with targets and inhibiting translation and/or inducing RNA degradation 4) There are three main classes: a) micro RNAs (miRNAs) b) small interfering RNAs (siRNAs) c) piwi-associated RNAs (piRNAs) 5) miRNAs and siRNAs differ in their biogenesis

Processing of pre-mRNA 3' end processing Tell me about alternative cleavage and polyadenylation

1) Some RNAs contain a single constitutive cleavage/polyadenylation signals (type I). Other RNAs contain several signals in the terminal exon (type II). Finally, some RNAs contain multiple signals in different exons (type III). In the last case, the choice of polyadenylation site is linked to alternative splicing 2) Below we consider one example of alternative polyadenylation (type II) a) Sex-Lethal (SXL) (BL: L for ladies) is a Drosophila RNA-binding protein expressed only in females b) SXL regulates splicing, translation and polyadenylation c) One target for polyadenylation regulation is an mRNA called enhancer of rudimentary or e(r) d) e(r) contains two polyadenylation sites; in males, the proximal site is used; in females, the distal is preferred e) Mechanism: In females, SXL binds to e(r) pre-mRNA and competes with CstF for binding to the proximal GU-rich element(GU is a signal to bind CstF for cleavage); this leads to the use of the next available site. By contrast, in males CstF can bind to the proximal site and promote its use f) Why is this important? The female-specific part of the 3' UTR contains sequences that induce translational repression; therefore, the e(r) protein is not produced in female flies

Splicing Regulation of alternative splicing: What determines if a splice site is used?

1) Strength of the splice sites (similarity of the 5' splice site, branch point, pyrimidine tract to the consensus sequence). When competing splice sites are present in a pre-mRNA the stronger one is used 2) Action of RNA-binding proteins binding to repressors or enhancers. These RBPs are expressed and/or activated in tissue-specific or developmentally controlled ways 3)RNA secondary structures, which can mask cis regulatory sequences

Initiation of translation in prokaryotes. The process of initiation

1) The 30S ribosomal subunit associates with IF3 and IF1 2) The 30S/IF1/IF3 complex interacts with the mRNA at the ribosome binding site 3) IF2 forms a ternary complex with GTP and charged initiator tRNA 4) The ternary complex joins 30S on the mRNA, forming the 30S initiation complex. Note that the initiator tRNA is at the P site (this is the only tRNA that enters the ribosome at this site) 5) The 50S subunit joins the initiation complex, leading to GTP hydrolysis by IF2 and to the release of all initiation factors. This is called the 70 S initiation complex 6) IF2 is recycled by exchanging GDP with GTP 7) As a result of this process, the 70S ribosome is assembled at the ribosome binding site with the initiator tRNA in the P site and an empty A site

Splicing Functions of alternative splicing - example 3: the Drosophila sex-lethal RNA

1) The Drosophila Sex-Lethal protein (SXL) is expressed in females, but not in males 2)In males, exon 3 included. This exon contains a stop codon, resulting in no SXL protein 3)In females, exon 3 is skipped, allowing the production of a full-length protein 4)Exons containing premature termination codons are often called 'poison exons' 5)In this case alternative splicing creates an on/off switch for gene expression

Splicing Regulation of alternative splicing - example 2: Transformer splicing by the SXL protein

1) The Transformer RNA contains two alternative 3' splice sites a) In males, the first one is used: the exon contains a premature termination codon (PTC) and no protein is produced b) In females, the second one is selected and the Transformer protein is produced 2) How is this regulated? a) The choice of alternative splice site is regulated by SXL, which is expressed only in females b) In males, no SXL proteins is present, and U2AF65 binds to the proximal site and selects it for splicing c) In females, SXL binds specifically to the proximal polypyrimidine tract (sequence UUUUUUUUCAG) but not to the distal one, preventing U2AF₆₅ from binding; U2AF₆₅ binds to and directs splicing to the distal site

Initiation of translation in prokaryotes. Initiator tRNA

1) The aminoacyl-tRNA used for translation initiation is called the initiator 2) The initiator is always charged with formyl methionine; thus, all bacterial proteins are synthesized with formyl methionine as their first aminoacid 3) The formyl group is rapidly lost by hydrolysis catalysed by a peptide deformylase 4) In about 50% of the proteins, the initial methionine is removed by a methionine aminopeptidase (this is a slower process)

RNA degradation Mechanisms of mRNA decay in the cytoplasm

1) The modifications at the 5' and 3' ends of mRNAs (cap, and polya(A) tail with bound poly(A) binding protein) protect them from degradation by exonucleases 2) Degradation usually starts by removing one (or both) of these protective structures, followed by digestion by exonucleases 3) Degradation can also (more rarely) be initiated by endonucleases 4) Usually the first step is the shortening of the poly(A) tails by poly(A) nucleases (also called deadenylases). These enzymes are exonucleases specific for poly(A) sequences. Several multiprotein complexes have this activity 5) Once the poly(A) tail is very short (~10 nt), two pathways can follow: Decapping (removal of the cap) and 5'->3' degradation, or 3'->5' degradation 6) In the first pathway a protein complex called Lsm binds to the short poly(A) tail and promotes decapping. The cap is removed by a heterodimer called decapping enzyme. The process produces an mRNA with a 5' monophosphate that is degraded by an exonuclease called XRN1 7) In the second pathway degradation is performed by a 3'->5' exonuclease called the exosome (a multiprotein complex). The exosome also has functions in the nucleus 8) Both pathways are partly redundant: if either of them is inactivated there are relatively small changes in gene expression (measured with microarrays) 9) Other pathways are probably more specialised. They include deadenylation-independent decapping, endonuclease-initiated degradation and addition of poly(U) to histone mRNAs 10) Finally, micro-RNAs can also mediate RNA decay

Processing of pre-mRNA 3' end processing Tell me about Structure and function of the poly(A) tail

1) The poly(A) tail is added in the nucleus and is not encoded in the DNA. Its initial length is ~240 nucleotides in mammals and 120 nucleotides in yeast. It gets shorter during transport to the cytoplasm, and as mRNA 'ages' in cytoplasm. The rate of shortening is transcript-specific (important for RNA decay, see later) 2) Functions of the poly(A) tail: Protects the mRNA from 3' exonucleases, and controls degradation rate of mRNAs. It is also necessary for translation initiation (see later)

Translation in eukaryotes Role of the poly(A) tail in translation

1) The poly(A) tails are coated by a cytoplasmic poly(A)-binding protein (PABP) 2) eIF4G interacts with PABP, causing the mRNA to adopt a circular conformation 3) This interaction stimulates translation

RNA degradation Kinetics of RNA degradation

1) The rate of degradation is defined by the half-life of the mRNA 2) mRNAs show a wide range of half-lives (transcript-specific) a) In mammalian cells half-lives range from <20 minutes to >48 hours b) In yeast cells they range from 3 to 300 minutes

Translation elongation in prokaryotes Ribosome translocation

1) The ribosome moves three nucleotides along the mRNA. This leads to the movement of the newly-formed peptidyl-tRNA to the P site, and to the release of the deacylated tRNA through the E site 2) The process of translocation requires EF-G, another monomeric GTPbinding protein with GTPase activity. EF-G-GTP can only bind to the ribosome when EF-Tu is not present. GTP hydrolysis is not required for binding (not inhibited by GMP-PCP), but stimulates ribosome translocation and is necessary for EF-G from the ribosome. The GTPase activity of EF-G is stimulated by binding to the ribosome

RNA degradation Importance of RNA decay

1) The steady state amounts of an mRNA are determined by the balance between transcription and RNA degradation. An important, often overlooked consequence of this, is that changes in mRNA levels can be achieved by modulating transcription rates, decay rates or both simultaneously 2) Quality control for the removal of defective RNAs

Eukaryotic small regulatory RNAs Biogenesis of miRNAs:

1) There are about ~500 different miRNAs in humans 2) miRNAs are transcribed by pol II as long precursors (pre-miRNAs) that contain local hairpins 3) They are cleaved by a nuclease called Drosha in the nucleus, which releases the hairpins 4) After export to the cytoplasm, they are cleaved further by Dicer, releasing the miRNA duplex (~22 nt) 5) The miRNA is then loaded onto a multiprotein complex called RISC (for RNA-induced silencing complex), which contains proteins of the argonaute family. During the loading process one strand is degraded (passenger strand) and another one is kept (guide/miRNA strand)

Splicing Tell me about Functions of alternative splicing - example 1: the SV40 T antigen

1) There is competition between two 5' splice sites a) If the first one is selected the mRNA encodes the large T antigen b) If the second one is chosen the mRNA encodes the small t antigen (the mRNA is longer, but the extended region in exon 1 contains a stop codon) 2) t is produced during early infection (represses apoptosis) and T later (induces transformation) 3) Alternative splicing creates two proteins with different functions

Splicing Functions of alternative splicing - example 2: the Drosophila DSCAM gene

1) This gene contains 24 exons: some of them are constitutive, while some others are chosen among a large number of mutually exclusive exons 2) The total number of possible isoforms is over 38,000 (note that the Drosophila genome only contains around 13,000 genes) 3) The DSCAM protein is required for neuronal patterning: in this case, alternative splicing creates protein variability for the recognition of specific neurons

What is the experimental outline that you did with POU?

1) Use PCR to amplify the DNA corresponding to the POU domain from a longer template, and incorporate a different restriction site at each end to allow directional cloning. 2) You will then cut the PCR product with a suitable restriction enzymes and ligate it into a vector for blue/white screening. 3) Then express the POU domain in E Coli. The expression plasmid is called pMAT8 and contains the DNA encoding the POU domain as a fusion protein with maltose binding protein (MBP) to aid solubility and a hexahistidine tag (His)₆ for purification. The fusion protein is under the control of a promoter recognized by the RNA polymerase from bacteriophage T7, whose expression is in turn switched on in the presence of lactose or its stable analogue isopropyl β-D-1-thiogalactopyranoside (IPTG) 4) Next you will purify the protein that you expressed previously, using an affinity column and in the final week you will use electrophoresis mobility shifts assays (EMSA) to characterize DNA binding by the POU domain.

Protein Purification using chromatography?

1) Using genetically introduced affinity tags, eg. the His-tag 2) By taking advantage of the charge properties of the protein in ion exhange chromatography 3) By using hydrophobic surfaces on the protein that can interact with hydrophobic interaction column 4) By separating proteins based on the size and shape in size exclusion chromatography

Sources where you can get sequence information for your PCR(for the primers I think)

1) You have the sequence already (you directly sequenced it?) 2) You might obtain the sequence directly using genomic data (use online resources to find your sequence) 3) You may know a bit of the amino acid sequence of a protein encoded by the DNA 4) If the DNA sequence were part of a family with conserved features (say encoding a set proteins with a conserved substrate binding site), you could design primers for those features.

Prokaryotic Transcription Mechanism Identification and characterization of promoters

1) consensus Sequences 2. Examine natural promoter mutations 3. Generate targeted mutations 4. Biochemical mapping of: a) DNA-protein interactions (following slides) b) DNA melting: KMnO₄ oxidation of single-stranded thymidines NB: 1-3 identify specific bases required for recognition Methods in 4a can identify more extensive regions of DNA in contact with protein - (sequence non-specific) Natural promoter mutations can be identified as mutations that affect the quantity but not the sequence of the mRNA. They can be classified as UP or DOWN mutations depending upon whether the mutation results in higher or lower levels of mRNA. For a strong promoter matching the consensus sequences well, DOWN mutations are more common. For weak promoters, UP mutations may also be possible. Mutations can also be generated experimentally, often guided by information from consensus sequences. Methods for biochemically mapping DNA-protein interactions are shown on the following slides. KMnO4 reacts preferentially with unpaired thymine bases, oxidizing the C5-C6 double bond, adding hydroxyl groups to C5 and C6. Subsequent alkali treatment cleaves the phosphodiester backbone at modified positions. Sensitivity to KMnO4 is therefore a useful biochemical tool for monitoring where DNA becomes unwound e.g. during transcription initiation.

Regulation of translation in eukaryotic cells Translational control by eIF2B phosphorylation

1) eIF2 is a trimeric GTP-binding protein 2) eIF2-GDP needs to be recycled to the GTP form before it can function, and recycling requires a guanine nucleotide exchange factors (GEF) called eIF2B 3) Phosphorylation of eIF2α sequesters eIF2B in complex with eIF2-GDP; as eIF2B is less abundant than eIF2, eIF2 cannot be recycled and translation initiation is blocked globally 4) Several kinases can phosphorylate eIF2 to downregulate translation, for example: a) Protein kinase R (PKR) is activated by dsRNA and is part of the antiviral response (it shuts down protein synthesis in infected cells) b) The PKR-like endoplasmic reticulum kinase (PERK), which is activated by the presence of unfolded/misfolded proteins in the ER

Translation Initiation of translation in prokaryotes. Factors required?

1) mRNA with a ribosome binding site 2) Ribosomes (small and large subunits) 3) Initiator tRNA: fMet-tRNAf (see below) 4) Three initiation factors: a) IF1: Binds to the A site of the ribosome and prevents access of tRNAs (you are initiating, its not elongation yet so its blocked) b) IF2: Forms a complex with the initiator tRNA and GTP (has GTPase activity) c) IF3: Inhibits 30S/50S reassociation (in initiation, you want these 2 to split apart so you can sandwich the mRNA). 5) GTP (but not ATP)

Hallmarks of enzyme catalysis?

1) occurs in an active site 2) saturation kinetics 3) specificity 4) large rate accelerations (makes them go faster)

Splicing Trans factors required for pre-mRNA splicing

1) snRNPs: small nuclear RiboNucleoProtein particles a) Contain a small nuclear RNA U1, U2, etc, associated with several proteins b) Involved in several RNA processing reactions c) The RNA component provides key function of snRNP (often by directly base-pairing with its substrate) d) U1, 2, 4, 5 and 6 involved in splicing e) U1 snRNA forms base pairs with the 5' splice site f) U2 snRNA forms base pairs with the branch point 2) U2AF (U2 snRNP Auxiliary Factor): Contains a large subunit (U2AF⁶⁵) that binds the polypyrimidine tract, and a small subunit (U2AF³⁵) that associates with the 3' splice site

How would you purify the restriction digested DNA using Qiaquick spin column?

1. Add 250 µl Buffer PB to the restriction reaction and mix. Check that the colour of the mixture is yellow. 2. Place a QIAquick spin column in a 2 ml collection tube. 3. Apply the sample to the QIAquick column and centrifuge for 60 sec (balance!). 4. Discard flow-through. Place the QIAquick column back into the same tube. 5. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 60 sec. 6. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the column for an additional 1 min. 7. Place the QIAquick column in a clean 1.5 ml microfuge tube. 8. To elute DNA, add 50 µl Buffer EB1 to the centre of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.

Prokaryotic Transcription Mechanism What are the 4 Methods to map DNA-protein interactions?

1. Electrophoretic mobility shift assay 2. Footprinting 3. Modification interference 4. Chromatin immunoprecipitation (ChIP)

Primer design You need to have some sequence information before you can do PCR, of course. This could come from a number of sources:

1. You might have the sequence already: § E.g. from a larger piece of DNA which you've sequenced and from which you want to amplify a smaller section - E.g. a cloned DNA 2. You may be able to obtain the sequence directly using genomic data (pp116-117) 3. You may know a bit of the amino acid sequence of a protein encoded by the DNA

How wide is the minor groove? How wide is the major groove? What is the width of DNA itself?

1.2 nm 2.2 nm 2 nm - The dimensions of major and minor grooves also vary

relaxed DNA bases per turn of the double helix

10.5 bases per turn

value of Kw?

10⁻¹⁴ M²

All bases found in nucleic acids have a characteristic absorption of light in the ultraviolet region, and nucleic acids have a maximum absorbance at around _________ What is the wavelength?

260nm

Tell me about Alternative Cloning Methods (without the use of PCR)

6.1 Sub-cloning from existing clone It is not always necessary to use PCR to amplify sequences to clone. It may be that you can simply use restriction enzymes to sub-clone sequences from an existing clone - if there are restriction sites in convenient locations. Sometimes MCSs are conserved between different plasmids (sometimes they may have the same origin), and in this case sub-cloning may be convenient. 6.2 Use of synthetic genes It is possible to order a synthetic gene - synthetic genes are open reading frame sequences from publicly annotated genes that are generated synthetically. This makes obtaining clones very simple, but is relatively expensive. Having said that, the cost is not prohibitive if there are no other options or if cloning is difficult for some reason. Synthetic genes are particularly useful for: • Genes for which no correct clones are available or hard to obtain otherwise (e.g. Full ORF clones, splice variants) • Increased expression yields due to the sequence optimization (when expressing your clone in your preferred expression system - E.g. E. coli, yeast) • Design of the sequence due to your specific requirements (E.g. introduction of point mutations, insertions or deletions, epitope and domain shuffling, introduction of fusion tags and particular restriction sites) A number of companies provide this service - E.g. Source BioScience.

What do we do once we have a clone? Tell me about performing in vitro assays

7.2 Performing in vitro assays There are various types of in vitro assays that you may want to perform using your favourite protein - E.g. binding assays (E.g. gel shift assays, filter binding assays), footprinting assays, UV crosslinking, editing assays, structural analyses etc. To carry out these types of assays, you may need various reagents - E.g. RNA probes, DNA probes, recombinant protein etc. Two methods that we will consider in more detail are: • Preparing in vitro transcribed RNA • Preparing recombinant protein. Brandon: you prepare the RNA and protein so you can assay them I think.

7.7 Making transgenic animals to analyze the function of your protein

7.7 Making transgenic animals to analyze the function of your protein Change the expression level of your favourite protein and then look at the phenotype of the resulting transgenic animal. Do this to work out function of protein. There are 2 main methods for generating transgenic whole organisms: A. Microinjection of DNA into the pronucleus of fertilized eggs at single cell stage B. Use of cultured embryonic stem (ES) cells

DNase footprinting assay

A DNase footprinting assay is a DNA footprinting technique from molecular biology/biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short), to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.

Give me a brief recap about restriction enzymes

A brief recap about restriction enzymes: These are found in bacteria, and were discovered by the fact that their presence restricted the ability of phages to grow in those bacteria. How? The restriction enzymes in E. coli cells degrade incoming phage DNA. In order to protect its own DNA from digestion by restriction enzymes, E. coli have a corresponding methylating enzyme that methylates its DNA at the restriction enzyme target sites - methylation commonly occurs at cytosine (C) and adenine (A) bases, and predominantly forms 5-methylcytosine (m5C), N4methylcytosine (m4C), and N6-methyladenine (m6A) derivatives. Methylation of E. coli DNA thus prevents it being degraded by their endogenous restriction enzymes. The attacking phage DNA is not methylated, so is subject to attack.

What is a bromodomain?

A bromodomain is an approximately 110 amino acid protein domain that recognizes acetylated lysine residues, such as those on the N-terminal tails of histones. Bromodomains, as the "readers" of lysine acetylation, are responsible in transducing the signal carried by acetylated lysine residues and translating it into various normal or abnormal phenotypes. Their affinity is higher for regions where multiple acetylation sites exist in proximity. This recognition is often a prerequisite for protein-histone association and chromatin remodeling. The domain itself adopts an all-α protein fold, a bundle of four alpha helices each separated by loop regions of variable lengths that form a hydrophobic pocket that recognizes the acetyl lysine.

What is a chaotropic agent?

A chaotropic agent is a molecule in water solution that can disrupt hydrogen bonding network between water molecules (ie exerts chaotropic activity). This has an effect on the stability of the native state of other molecules in the solution, main macromolecules (proteins, nucleic acids) by weakening the hydrophobic effect. Ethanol and Guanidinium chloride are common chaotropic agents. Magnesium chloride, SDS and urea are also common chaotropic agents.

What are expressed sequence tags?

A collection of cloned cDNAs, where the 5' or 3' ends of the sequence are determined. ESTs are typically >400nt in length and may be prepared from different tissues/developmental stages. Many EST databases are commerically available EST is a short sub-sequence of a cDNA sequence ESTs may be used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination. Brandon: they are called "expressed" because they are expressed as mRNA in the cell.

Filter binding assay?

A filter binding assay is a simple way to quickly study many samples. It measures affinities between two molecules (often protein and DNA) using a filter. The filter is constructed of nitrocellulose paper, which is negatively charged. Since most proteins have a net positive charge, nitrocellulose paper is ideal for immobilizing proteins. DNA is negatively charged due to the phosphate backbone and will not "stick" to the nitrocellulose on its own, however, any DNA that has been bound by protein will stick. The exact amount of DNA "stuck" to the nitrocellulose is quantified by measuring the amount of radioactivity on the filter using a scintillation counter.

What is a gene cassette?

A gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tend to be very small; on the order of 500-1000 base pairs. They may exist incorporated into an integron or freely as circular DNA. Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer. These cassettes often carry antibiotic resistance genes. An example would be the kanMX cassette which confers kanamycin (an antibiotic) resistance upon bacteria.

Tell me about northern blot

A general blotting procedure starts with extraction of total RNA from a homogenized tissue sample or from cells. Eukaryotic mRNA can then be isolated through the use of oligo (dT) cellulose chromatography to isolate only those RNAs with a poly(A) tail. RNA samples are then separated by gel electrophoresis. Since the gels are fragile and the probes are unable to enter the matrix, the RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. The transfer buffer used for the blotting usually contains formamide because it lowers(mistake? You want the RNA to be separated, so increases annealing temp, or lower the denaturation temperature) the annealing temperature of the probe-RNA interaction, thus eliminating the need for high temperatures, which could cause RNA degradation. Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat. After a probe has been labeled, it is hybridized to the RNA on the membrane. Experimental conditions that can affect the efficiency and specificity of hybridization include ionic strength, viscosity, duplex length, mismatched base pairs, and base composition. The membrane is washed to ensure that the probe has bound specifically and to prevent background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. To create controls for comparison in a northern blot, samples not displaying the gene product of interest can be used after determination by microarrays or RT-PCR.

Polypyrimidine Tract, what binds to it?

A number of protein factors bind to or associate with the polypyrimidine tract, including the spliceosome component U2AF and the polypyrimidine tract-binding protein (PTB), which plays a regulatory role in alternative splicing. PTB's primary function is in exon silencing, by which a particular exon region normally spliced into the mature mRNA is instead left out, resulting in the expression of an isoform of the protein for which the mRNA codes. Because PTB is ubiquitously expressed in many higher eukaryotes, it is thought to suppress the inclusion of "weak" exons with poorly defined splice sites.[2] However, PTB binding is not sufficient to suppress "robust" exons

Reporter Genes

A reporter gene is a gene attached to a regulatory sequence of another gene of interest - reporter genes are easily identified and measured. For example, we could use a reporter gene to see whereabouts in an organism a protein of interest ends up or we could determine how active a particular promoter is in different tissues (it may not be possible to measure the amount of the protein whose gene is under the control of the promoter directly) • So we could attach a "reporter gene" to the promoter belonging to the protein of interest. This is a gene whose product is easy to measure in a simple assay. • The construct would then be introduced into the relevant tissues, and expression would be visualized. • You could also measure the activity of the reporter gene product. Making certain assumptions (what are they?) this gives you an estimate of the relative activity of the promoter.

RNA degradation Quantification of RNA half-lives

A simple method is the following: 1) Block transcription of the gene: in the absence of transcription, changes in mRNA levels reflect only degradation 2) Follow changes in mRNA levels in a time course, then use the information to calculate the half-life 3) Transcription can be blocked in several ways: a) Treat cells with inhibitors of RNA Pol II b) Use thermosensitive mutants in components of RNA polymerase II (available in yeast) c) Clone the gene of interest under the control of a regulatable promoter (a promoter that can be switched on/off)

Initiation of translation in prokaryotes. Selection of the correct site for initiation Tell me an experiment to isolate and identify translation initiation sites

A typical mRNA contains multiple AUG codons (also, less frequently, translation can be initiated from GUG or UGG). How does the 30S subunit find the correct initiation codon? The experiment below allows the isolation and identification of translation initiation sites: The protected fragments contain two common features: 1) The initiation codon (AUG, less commonly GUG/UUG) 2) A polypurine stretch about 10 nucleotides upstream of the initiation codon called the ShineDalgarno sequence or ribosome-binding site (RBS). The consensus sequence is AGGAGG a) The Shine-Dalgarno sequence is complementary to a sequence in the 3' end of the 16S rRNA b) The degree of complementarity varies among transcripts from 4 to 9 bases The model is that the Shine-Dalgarno sequence pairs with the complementary region in the 16S rRNA, allowing the identification of the initiation codon. In addition, the fMet-tRNA anticodon interacts with the AUG. This model is supported by in vitro and in vivo evidence

What is the affinity Tag?

Affinity tags are appended to proteins so that they can be purified from their crude biological source using an affinity technique These include chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag and glutathione-S-transferase (GST). The poly(His) tag is a widely used protein tag, which binds to metal matrices.

Splicing Regulation of splicing by RNA-binding proteins Splicing Activators

Activate the use of splice sites. Example: SR proteins a) Modular structure: contain an RNA-binding domain (one or two RRMs or RNA-recognition motifs) and a protein interaction domain (RS domain). Note other proteins (such as U2AF65 and U2AF35) have RS domains b) SR proteins can activate splicing by several mechanisms. For example, some SR proteins promote the recruitment of U2AF to splice sites through the interactions between the RS domain of the SR protein and that of U2AF35

Splicing The Spliceosome

Analysis of in vitro splicing reactions on non-denaturing electrophoretic gels or on a glycerol gradient shows that the pre-mRNA becomes part of a large ribonucleoprotein complex (60S), called the spliceosome. The assembly of the spliceosome requires ATP. The spliceosome contains the U1, U2, U5 and U4/6 snRNPs, as well as many other proteins. Some of these factors are involved in specific recognition of the splice site consensus sequences. In particular, snRNA-pre-mRNA base pairing is crucial. The spliceosome is assembled following an ordered pathway (but, unlike ribosomal subunits, the spliceosome only fully assembles on a pre-mRNA). The conversion of the B complex into a catalytic spliceosome involves extensive rearrangements of the U snRNAs: 1) The pairing between U4:U6 is broken 2) U1 is displaced from the 5' splice site 3) U6 base-pairs with the 5' splice site and with U2 snRNA As a result, a network of RNA-RNA interactions among the 5'SS-U6-U2-branch point aligns the substrate pre-mRNA for the first catalytic step and generates the catalytic site RNA splicing is thought to be an RNA catalysed reaction, with the catalytic core of the spliceosome formed by U2 and U6 snRNAs (and possibly some proteins). Some protein splicing factors are members of a family of ATP-dependent RNA helicases. These may catalyze some of the RNA conformational changes, such as the U4-U6 to U2-U6 rearrangement.

Chromatin Packaging Nucleosome positioning

Arginines bind more tightly to narrow minor grooves, because narrow, much improved electrostatic interaction between negative phosphates and positive arginines.

Genome-wide approaches to study gene expression

As discussed in the first lecture, the use of DNA microarrays or high throughput sequencing can provide a 'global' view of gene expression control. We will briefly discuss how these methods can be applied to the study of translation and splicing. 1) Translational control: a genome-wide view 2) A genome-wide view of splicing

Methods to study posttranscriptional processes Tell me about Identification of cis-acting sequences

As for DNA, consensus sequences can be identified by comparing multiple sequences and their importance tested by mutational analysis. They can also be identified by isolating and characterising RNAs bound to specific proteins

Methods to study posttranscriptional processes

As is the case for transcription, posttranscriptional processes are controlled by cis-acting sequences (in this case in RNA molecules) that are recognised by trans-acting factors (proteins or RNA molecules). Many approaches used to study transcriptional and posttranscriptional control are very similar and will be discussed in Prof Luisi's lectures (see also Dr Scadden's lectures). 1) Identification of cis-acting sequences 2) Mapping RNA-protein interactions 3) Single-gene view and genome-wide analysis

Regulation of translational efficiency in bacteria Attenuation

Attenuation is a mechanism to repress gene expression based on premature termination of transcription. Attenuation is often linked to translation, and thus will be discussed in this section. Bacteria used attenuation to couple the expression of amino acid biosynthetic operons to the availability of the corresponding amino-acyl tRNA. Attenuation relies on the following features of prokaryotic gene expression: 1) Transcription and translation are coupled 2) mRNA folding is cotranscriptional (i.e., the mRNA forms secondary structures before transcription is complete) 3) mRNAs can form alternative, mutually exclusive secondary structures 4) We will use the tryptophan (trp) operon as an example. The trp mRNA contains a control region at the 5' end, followed by genes encoding five structural genes 5) If tryptophan is scarce (measured as low tryptophan-tRNA), the whole operon is transcribed 6) If tryptophan is abundant, transcription terminates prematurely before the first structural gene Attenuation is controlled by cis-acting elements in the control regions: 1) 5' mini-cistron containing two consecutive trp codons Met Lys Ala Ile Phe Val Leu Lys Gly *Trp* *Trp* Arg Thr Ser (STOP) 2) Four potential stem-loop-forming elements with mutually exclusive conformations: (2:3 ) or (1:2 and 3:4) 3) Element 1 overlaps with the mini-cistron 4) Elements 3 and 4 are followed by a poly(U) track and can form a ρ-independent terminator The system works as follows: 1) In the presence of trp the ribosome translates the mini-cistron and favours the 3:4 structure; transcription stops prematurely 2) If trp is scarce, the ribosome gets stalled at the trp codons; the 2:3 structure forms and prevents the formation of the terminator, allowing transcription of the whole gene

Most common DNA helix type in vivo?

B-form helix

The original DNA fibre diffraction patterns (Rosalind Franklin) showed two forms, what are they? Biological relevance of Z-form DNA?

B-form in high humidity A-form in low humidity A third form — referred to as Z because of the zig-zag trajectory of the sugarphosphate backbones (slide 5 shows this rather clearly) — is a left-handed helix and is favoured by alternating purine-pyrimidine repeats. Its biological relevance is unclear. It is stabilized by "Z-binding" proteins & might be more relevant in RNA. Proteins with Z-binding domains are often RNA binding proteins.

Regulation of translational efficiency in bacteria Termination of transcription in bacteria

Before we move on to the study of attenuation we need an introduction to transcriptional termination in bacteria: 1) Termination is triggered by signals present in the RNA that is being transcribed, leading to the dissociation of RNA polymerase from its template and the release of the RNA 2) Two types of terminator have been identified in E. coli: Rho-independent and Rho-dependent 3) Rho-independent: a) There are no factors involved other than the RNA signal b) Transcripts have two structural features at the 3' end: i)G-C rich hairpin structure of variable length ii) Run of ~6 U residues 4) Both features are required for termination 5) Termination is disrupted by: a) Mutations that destabilize the stem b) Mutations that stabilize DNA-RNA hybrid after the stemloop c) Deletion of poly(U) d) In vitro transcription with ITP in place of GTP (I=C pairs only form two hydrogen bonds) 6) Termination occurs because: a) The hairpin in the nascent RNA causes 'pausing' of polymerase b) The base pairing between the rU and dA tracts is particularly weak, allowing the RNA and template DNA to dissociate Rho-dependent: a) Requires a protein called rho, which forms a ringshaped hexamer b) Rho has RNA-dependent ATPase activity and may act as a helicase c) The cis-acting elements recognized by rho are less well defined than those of rho-independent terminators: A C-rich region followed by a hairpin that induces transcriptional pausing d) Model: rho binds to the C-rich region, and translocates along the RNA until it reaches RNAP, where it induces its dissociation. The hairpin delays RNAP, allowing rho to reach it

Yeast 2 Hybrid What proteins might give rise to false positives or negatives?

Broadly, there are two kinds of false positive - those that are caused by protein-protein interactions that wouldn't normally happen, and those that don't require any protein-protein interaction at all. If the bait protein happens to have a hydrophobic patch exposed, other hydrophobic proteins might well stick by chance. Alternatively, the genuine target might have relatives in the library (i.e. it might be part of a multigene family) that don't usually bind to the bait, but can bind sufficiently to set off the reporter. A protein that was a general transcription activator might set off the reporter without needing to bind to the bait at all. It's also worth thinking why you might fail to see a genuine interaction. There might be steric effects - the fusion of the bait to the DNA-binding domain might block the region of the bait that's needed for interaction with the target. Also, the interacting proteins need to be located in the nucleus, so if the fusions can't be imported into the nucleus, although the interaction might take place, it won't set off the reporter gene. Remember, too that many important protein-protein interactions are transient, so the binding may not last long enough to set off the reporter.

Prokaryotic Transcription Mechanism Methods to map DNA-protein interactions 4. Chromatin immunoprecipitation (ChIP)

CH₂NH[CH₂]₄CαH ChIP has been used very extensively in eukaryotes. In contrast to the preceding methods, the resolution is much lower (~100-200 bp). It is a method that is used to probe DNA binding in vivo - most of the other methods are applicable only in vitro (exception: footprinting using DMS can be in vivo, although without the use of a radiolabelled DNA probe)

Processing of pre-mRNA 5' end processing: capping Tell me about Capping specificity

Capping takes place cotranscriptionally and is performed by enzymes associated with Pol II CTD. As this structure does not exist in Pol I and Pol III, transcripts produced by those enzymes are not capped. In addition, Pol II CTD allosterically activates mammalian RNA guanylyl transferase. Evidence: 1) Pol II CTD is required for capping a) Cells are transfected with one of two versions of amanitinresistant RNA Pol II (amanitin is an inhibitor of Pol II) b) One version contains a normal CTD with 52 repeats (WT), while the other contains only 5 repeats (∆CTD) c) Endogenous RNA Pol II is inhibited with amanitin, cells are incubated for 24 hours, and capped and uncapped mRNAs are separated and quantified d) Result: the fraction of capped mRNAs is much lower in ∆CTD than in the wild type, indicating that the CTD is required for capping BL: does this experiment suggest that alpha amanitin deactivates the CTD of Pol II somehow, since the numbers seem identical? Not necessarily, but its some evidence. 2) Capping enzymes associate with phosphorylated Pol II CTD: a) Experiment 1: i) Prepare nuclear extract ii) Pass extract through one of three affinity columns: Wild type CTD / mutant CTD / phosphorylated CTD iii) Measure capping activity in sample retained in each column iv) Result: capping activity is retained only in the column containing phosphorylated CTD b) Experiment 2: i) Performed in fission yeast (S. pombe) ii) Make mutant CTD that cannot be phosphorylated at S5 (in all repeats) YSPT*S*PS-> YSPT*A*PS iii) Replace endogenous gene with mutated form -> cells do not grow iv) Fuse mammalian capping enzyme to the CTD -> cells with the mutation can now grow

Immunoprecipitations

Cell lysate containing complex mixture of proteins, you only want one. You can use immunoprecipitation assays to determine which proteins interact with your protein of interest. • You need antibody that will specifically recognize your protein - o get antibody that will recognize native (untagged) version of protein of interest. antibodies available commercially or from other labs o important to verify that antibody only recognizes protein of interest. not all antibodies are good for immunoprecipitation experiments - and you don't know until you try. o Alternatively, you may make use of antibodies that recognize epitope tags (E.g. Flag, Myc, HA etc) - in this case need to overexpress a tagged-version of protein of interest. Use of antibodies that recognize epitope tags is often an advantage as they typically immunoprecipitate tagged proteins very efficiently • Antibody-protein complexes (immunoprecipitates) are subsequently captured using protein A or protein G beads - these may be magnetic beads, so complexes can be isolated using a magnet. • Immunoprecipitates are analyzed on SDS-PAGE - proteins co-precipitating with your protein of interest can be identified using mass spectrometry (E.g. Cambridge Centre for Proteomics (http://proteomics.bio.cam.ac.uk/core-facility/ccp-brochure-2013/view)) • Immunoblots (Westerns) can subsequently be used to verify protein-protein interactions.

Chloramphenicol

Chloramphenicol is extremely lipid-soluble; it remains relatively unbound to protein and is a small molecule. It has a large apparent volume of distribution and penetrates effectively into all tissues of the body, including the brain. Distribution is not uniform, with highest concentrations found in the liver and kidney, with lowest in the brain and cerebrospinal fluid. The concentration achieved in brain and cerebrospinal fluid is around 30 to 50% of the overall average body concentration, even when the meninges are not inflamed; this increases to as high as 89% when the meninges are inflamed. Chloramphenicol increases the absorption of iron. Chloramphenicol passes into breast milk, so should therefore be avoided during breast feeding, if possible Chloramphenicol is antagonistic with most cephalosporins and using both together should be avoided in the treatment of infections Chloramphenicol is a bacteriostatic by inhibiting protein synthesis. It prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome. It specifically binds to A2451 and A2452 residues in the 23S rRNA of the 50S ribosomal subunit, preventing peptide bond formation. While chloramphenicol and the macrolide class of antibiotics both interact with ribosomes, chloramphenicol is not a macrolide. It directly interferes with substrate binding, whereas macrolides sterically block the progression of the growing peptide.

Eukaryotic transcription - activating transcription factors and enhancers C3 shows looping between β-Globin Locus Control Region and active globin gene promoters

Chromatin conformation capture (C3) and variants of the method are increasingly being used to investigate long-range chromatin interactions. The preceding two slides use qPCR to investigate suspected long-range looping interactions between active β globin gene promoters and the distant "locus control regions", which act like enhancers. The C3 interactions were only observed in cell types (e.g. liver) where the genes are switched on, but not in non-expressing cells (e.g. brain). Thus, there is a correlation between gene activity and long-range chromatin loops. But these experiments required prior knowledge about the important DNA elements (promoters, locus control regions). Increasingly, instead of qPCR (which requires prior knowledge and selection of primer sequences for PCR), the final ligated products are subjected to deepsequencing to give a genome-wide view of long-range chromatin interactions (referred to as C4). This allows previously unsuspected interactions to be discovered, and has been used to generate 3D models of chromatin organization in the nuclei of single cells.

Tell me about CPSF

Cleavage and polyadenylation specificity factor (CPSF) is involved in the cleavage of the 3' signaling region from a newly synthesized pre-messenger RNA (pre-mRNA) molecule in the process of gene transcription. It is the first protein to bind to the signaling region near the cleavage site of the pre-mRNA, to which the poly(A) tail will be added by polynucleotide adenylyltransferase. The upstream signaling region has the canonical nucleotide sequence AAUAAA, which is highly conserved across the vast majority of pre-mRNAs. A second downstream signaling region, located on the portion of the pre-mRNA that is cleaved before polyadenylation, consists of a GU-rich region required for efficient processing.

Tell me about 'TA cloning'

Cloning PCR products into a vector.... This method takes advantage of the fact that Taq DNA polymerase has a terminal transferase activity that adds a non-templated A residue to the 3' end of PCR products. Taq can also be used to add a 3' A residue to blunt PCR products generated using high fidelity DNA polymerases (after the completion of the PCR). The PCR products with the A residue at the 3' end can subsequently be cloned into a commercially available vector that has a single complementary T-overhang (E.g. pGEM-T Easy (Promega)). The cloning is efficient, but has the disadvantage that cloning is non-directional. Brandon: so basically blunt ends become sticky ends so inserting into vector becomes easier

Initiation of translation in prokaryotes. In vivo evidence for base pairing of SD sequence and 16S rRNA

Compensatory mutations in 16S rRNA suppress a mutant SD sequence: 1) A plasmid containing the following two genes is constructed: a) The human growth hormone (hGH), under a constitutive promoter and containing a mutated SD box b) The E. coli 16S pre-rRNA gene, under the control of a heat-shock promoter, and containing a mutation that renders it resistant to spectinomycin, and a mutation in the SD-binding sequence that makes it complementary to the mutated SD box of the hGH gene Results: a) At 37°C no hGH is produced, indicating that the mutated SD sequence is not functional b) At 42°C hGH is produced, showing that the mutant 16 rRNA suppresses the mutation in the hGH SD box c) The addition of spectinomycin confirms that the suppression is specifically due to the expression of the mutated 16S rRNA Note that this initiation mechanism allows the recognition of multiple ribosome binding sites on a single mRNA, and thus the use of polycistronic mRNAs.

Synovial Fluid

Composed of Hyaluronan, keratan sulfate, chondroitin 6 sulfate is a viscous, non-Newtonian fluid found in the cavities of synovial joints

What is cyanine ?

Cyanine is the non-systematic name of a synthetic dye family belonging to polymethine group. The word cyanin is from the English word "cyan", which conventionally means a shade of blue-green There are three types of cyanines: Streptocyanines or open chain cyanines: R2N+=CH[CH=CH]n-NR2 (I) Hemicyanines: Aryl=N+=CH[CH=CH]n-NR2 (II) Closed chain cyanines: Aryl=N+=CH[CH=CH]n-N=Aryl (III)

A-form is difficult to recognise sequence-specifically Why?

DNA usually adopts the B-form. Importantly, the major groove is wide and accessible - so the information there (slides 12-13) can be "read" by proteins A-form is fatter, with 11bp/turn and a narrow, deep major groove, and the basepairs tilted. A "hole" in the middle of the helical axis viewed from above indicates the deep major groove. The deep NARROW major groove is not readily accessible for recognition by proteins. Relevance: dsRNA adopts A-form. Very difficult to recognise fully double-stranded Watson-Crick base-paired RNA in a sequence-specific manner. A-form dsRNA is often sensed as "foreign" e.g. virally derived (see Juan Mata's following lectures).

Internal initiation Evidence for internal initiation:

Dicistronic reporter: Insertion of part of the viral 5' UTR between two cistrons (containing the IRES) can drive internal initiation of the downstream cistron

Genome-wide approaches to study gene expression Translational control: a genome-wide view How to measure Translational efficiency

Different mRNAs are translated at different rates, and translational efficiency can be regulated by environmental or developmental changes. To obtain a genome-wide view of this regulation it is necessary to measure translation rates for every mRNA in the cell. This can be done using a method called ribosomal profiling. The method is based on the fact that mRNAs can be translated simultaneously by several ribosomes, and that the number of ribosomes associated with a transcript provides an estimate of how efficiently it is being translated. The method works as follows: 1) mRNAs associated with ribosomes (polysomes) are purified 2) The sample is treated with a ribonuclease that will degrade all the mRNA except those fragments 'protected' by the binding of the ribosome 3) The protected fragments are isolated and analysed by high throughput sequencing 4) The sequences are mapped to the genome and quantified 5) The number of protected sequences corresponding to an mRNA (normalised by the abundance of the mRNA) reflects how many ribosomes are bound to it, and hence how efficiently the mRNA is being translated

What is ADAR?

Double-stranded RNA-specific adenosine deaminase is an enzyme that in humans is encoded by the ADAR gene (which stands for adenosine deaminase acting on RNA). Adenosine deaminases acting on RNA (ADAR) are enzymes responsible for binding to double stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) by deamination. ADAR protein is a RNA-binding protein, which functions in RNA-editing through post-transcriptional modification of mRNA transcripts by changing the nucleotide content of the RNA.The conversion from A to I in the RNA disrupt the normal A:U pairing which makes the RNA unstable. Inosine is structurally similar to that of guanine (G) which leads to I to cytosine (C) binding. In RNA I functions the same as G in both translation and replication. Codon changes can arise from editing which may lead to changes in the coding sequences for proteins and their functions

What about DNA ligase? Where do we get that from?

E. coli has DNA ligase, which is used for routine DNA replication, repair etc. So we can use that. Many phages, such as T4, encode ligases, and we can also use those. T4 DNA ligase is the most routinely used one, but that won't ligate blunt ends. We need E. coli DNA ligase for that. Some other enzymes have ligase activity, such as topoisomerases, and these can also be used (often referred to as topo-cloning).

Translation elongation in prokaryotes Recycling of elongation factors

EF-G•GDP and EF-Tu•GDP must be recycled to their GTP-bound forms before they can take part in a new round of elongation 1) Recycling of EF-G does not require ancillary factors (it has higher affinity for GTP than GDP) 2) Recycling of EF-Tu needs the action of EF-Ts a) EF-Ts bind to EF-Tu and displaces GDP b) GTP displaces EF-Ts and binds to EF-Tu c) EF-Tu•GTP binds to aa-tRNA

Tell me about use of cultured embryonic stem (ES) cells when making transgenic animals to analyze the function of your protein

ES cells undergo manipulation and are re-injected into developing embryo. Some of the cells get incorporated into the embryo, which grows to form a mosaic. This approach is often used to knockout endogenous genes. So to make a knockout mouse, look at picture. In this case, the targeted gene is replaced by a positive selection marker, which is an antibiotic resistance gene (neomycin). This approach could also be used to make subtle changes (E.g. a point mutation) in the targeted gene. In addition, tissue-specific knockouts can be generated using the Cre system(another card)

Tell me about EF-Tu

Elongation factor thermo unstable - prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes Outside of the ribosome, EF-Tu complexed with GTP (EF-Tu • GTP) complexes with aa-tRNA to form a stable EF-Tu • GTP • aa-tRNA ternary complex. The binding of an aa-tRNA to EF-Tu • GTP allows for the ternary complex to be translocated to the A-site of an active ribosome, in which the anticodon of the tRNA binds to the codon of the mRNA. If the correct anticodon binds to the mRNA codon, the ribosome changes configuration and alters the geometry of the GTPase domain of EF-Tu, resulting in the hydrolysis of the GTP associated with the EF-Tu to GDP and Pi. As such, the ribosome functions as a GTPase-activating protein (GAP) for EF-Tu. Upon GTP hydrolysis, the conformation of EF-Tu changes drastically and dissociates from the aa-tRNA and ribosome complex The aa-tRNA then fully enters the A-site, where its amino acid is brought near the P-site's polypeptide and the ribosome catalyzes the covalent transfer of the polypeptide onto the amino acid In the cytoplasm, the deactivated EF-Tu • GDP is acted on by the prokaryotic elongation factor EF-Ts, which causes EF-Tu to release its bound GDP. Upon dissociation of EF-Ts, EF-Tu is able to complex with a GTP due to the 5- to 10-fold higher concentration of GTP than GDP in the cytoplasm, resulting in reactivated EF-Tu • GTP, which can then associate with another aa-tRNA

Eukaryotic transcription - activating transcription factors and enhancers Enhancers

Enhancers were originally identified in viral genomes as cis elements that can very strongly activate transcription, even though they can be located at great distances from their target promoters. The "enhancer trap" assay tests for potential enhancers by cloning suspected genomic DNA fragments downstream of a test gene in both orientations.

Translation in eukaryotes Initiation in eukaryotes: the scanning model What is the evidence for the scanning model?

Evidence for the scanning model: 1) >90% of mRNAs initiate at first AUG 2) Initiation at new AUGs inserted between cap and authentic AUG 3) Eukaryotic ribosomes do not bind to circularized mRNA (prokaryotic ones do) 4) Uncapped mRNA are translated accurately but inefficiently 5) Addition of m7G5'ppp5'G cap analogue reduces translation efficiency 6) Insertion of stable secondary structure between cap and AUG inhibits translation

What are salt bridges?

Example of salt bridge between amino acids glutamic acid and lysine demonstrating electrostatic interaction and hydrogen bonding The salt bridge most often arises from the anionic carboxylate (RCOO−) of either aspartic acid or glutamic acid and the cationic ammonium (RNH3+) from lysine or the guanidinium (RNHC(NH2)2+) of arginine (Figure 2).[1] Although these are the most common, other residues with ionizable side chains such as histidine, tyrosine, and serine can also participate, depending on outside factors perturbing their pKa's. The distance between the residues participating in the salt bridge is also cited as being important. The distance required is less than 4 Å (400 pm). Amino acids greater than this distance apart do not qualify as forming a salt bridge.[3] Due to the numerous ionizable side chains of amino acids found throughout a protein, the pH at which a protein is placed is crucial to its stability.

Protein expression, why is induction of IPTG carefully timed?

Expressing a foreign protein in large quantities is quite a strain for a bacterial cell and it is common for cell division to slow down once protein expression has been induced. For this reason, induction by IPTG is carefully timed, so that the bacterial cultures are in late log phase, that is, they are growing quickly but that they have not yet run out of nutrients in the medium. At this stage, there will be plenty of cells in the culture to produce protein.

Prokaryotic Transcription Mechanism Methods to map DNA-protein interactions 2. Footprinting

Footprinting and chemical modification interference These approaches provide single nucleotide resolution analysis of DNA-protein interactions in complementary ways. Footprinting asks at which positions on DNA does protein binding protect it from attack, while modification-interference asks at which positions does prior chemical modification subsequently prevent protein binding. For each of the chemical modications, a specific subsequent treatment leads to cleavage of the DNA at the modified position. For example, after ethylation of phosphates by ENU, the DNA can be cleaved only at the modified position by incubation at high pH (~13). Both of these approaches can identify contacts that extend beyond the bases that are specifically recognised - especially for an agent like ENU that interferes with phosphate backbone interactions.

Prokaryotic Transcription Mechanism Methods to map DNA-protein interactions 3. Modification interference

Footprinting and chemical modification interference These approaches provide single nucleotide resolution analysis of DNA-protein interactions in complementary ways. Footprinting asks at which positions on DNA does protein binding protect it from attack, while modification-interference asks at which positions does prior chemical modification subsequently prevent protein binding. For each of the chemical modications, a specific subsequent treatment leads to cleavage of the DNA at the modified position. For example, after ethylation of phosphates by ENU, the DNA can be cleaved only at the modified position by incubation at high pH (~13). Both of these approaches can identify contacts that extend beyond the bases that are specifically recognised - especially for an agent like ENU that interferes with phosphate backbone interactions.

Investigating Mechanisms of Gene Expression Genomic or cDNA Clone?

For eukaryotes, the choice of genomic or cDNA clone is important depending on the process being investigated. To investigate transcription or pre-mRNA processing, a genomic clone would be needed, since many of the relevant regulatory elements either do not get transcribed (e.g. promoter elements) or they are removed during RNA processing (e.g. splice sites in introns). On the other hand a genomic clone would be less useful for studying functions of mRNA e.g. by an in vitro translation assay - because it has introns - the open reading frame (ORF) has not yet been generated by splicing cDNAs are derived from mRNAs. They lack promoters, introns, but have additional features such as the poly A tail (or at least part of it - usually derived by using an oligo dT primer for reverse transcription). cDNAs can be used to investigate mRNA functions such as translation and mRNA stability. They can also be used to express eukaryotic proteins in E coli (genomic clone would be no good, as E coli does not have splicing machinery). However, increasingly, it is common to design completely synthetic genes (e.g. with optimized codons matching the host cells tRNA population) for expression of proteins in foreign organisms.

Tell me about porous medium of aqueous polyacrylamide

For large molecules like proteins (whose Mr values can lie between 1 kDa and 1 MDa), a porous medium of aqueous polyacrylamide is used. Polyacrylamide gels are prepared by dissolving acrylamide monomer, [CH₂=CH-CO-NH₂] and a cross linking agent called N,N'-methylenebis-(acrylamide), [(CH₂=CH-CO-NH-)₂CH₂] In the presence of a suitable catalyst, gels are formed, consisting of a three dimensional network of long chains of [-CH₂-CH₂(-CO-NH₂)]n cross-linked at intervals by methylene groups The pore size of the gel can be modified by varying the acrylamide monomer concentration. For example, gels containing 5% acrylamide will separate polypeptides of 100-400 kDa Mr while gels containing 15% acrylamide separate polypeptides of 10-100 kDa Mr

When would you use a porous medium of aqueous polyacrylamide

For large molecules like proteins (whose Mr values can lie between 1 kDa and 1 MDa), a porous medium of aqueous polyacrylamide is used.

Tell me about fusion with a 6x His-Tag

Fusion proteins for protein purification Addition of a run of six histidine residues is a useful tag (a 'His-tag') - and relatively small compared to some other tags At high pH, histidine sidechains chelate to metal ions, such as Ni²⁺ (why?). Ni²⁺ ions are immobilized on Sepharose, which is an inert, cross-linked polysaccharide polymer in the form of a small column. Expression of the fusion protein is induced in E. coli, and crude lysates containing the fusion protein (and millions of others) are then prepared. • The resulting protein can be purified with columns containing immobilized metal cations - E.g. nickel chelated to nitrilo-triacetic acid (NTA). The column is washed extensively to remove unbound proteins. The His-tagged protein of interest is stuck to your column • The protein is eluted from the column in one of two ways: o If your His-tagged protein has a protease cleavage site: for fusion proteins where the junction contains a protease recognition site (see above), the appropriate protease is added to the column and the protein is released by cleavage. o For His-tagged proteins lacking a cleavage site: the protein is eluted with something that competes for binding to the column. E.g. imidazole for the his-tagged proteins. The imidazole binds to the nickel (Ni²⁺) column and displaces the fusion protein.

GFP Tagged proteins

GFP used in analyzing the localization of proteins.... • Insert gene of interest into MCS to make GFP-fusion protein • Many variants of GFP (E.g. mCherry, Emerald, Venus) - makes it possible to visualize multiple proteins simultaneously • GFP-fusion protein can be visualized directly (fluorescence) or used for antibody recognition in immunoprecipitations, immunoblots (Westerns) etc. Example: Fluorescence microscopy is used to visualize mCherry-tagged TIAR (red) and GFP-tagged ADAR1 (green) simultaneously. The merged image is yellow, which enables you to see where the proteins co-localize.

Prokaryotic Transcription Mechanism Methods to map DNA-protein interactions 1. Electrophoretic mobility shift assay (EMSA, or "gel-shift" assay)

Gel-shift initially addresses whether a fragment of DNA binds to protein in vitro. The assay can use a purified protein, or a protein present in a cell extract To address the sequence specificity of interaction by gel shift, you either need to use labelled probes with altered sequence (mutations), or compare the ability of wild-type and mutant unlabelled DNAs to compete for protein binding (NB the "wedge" shapes above the gel on the right hand side represent increasing concentrations of competitor DNA). Effective competitors cause disappearance of the labelled complex band (a complex is still there in the gel, but containing the unlabelled DNA). Variants of the gel shift assay include the "supershift" e.g. if the DNA probe is incubated with a cell extract and you think you know the identity of the binding protein, you can test using antibodies to the candidate protein. The DNA-protein-antibody complex typically migrates even slower than the DNA-protein complex. In the case shown, antibodies against the diamond or hexagon shaped proteins would not supershift, but against the circular protein they would. EMSA, footprinting and modification interference (next 2 slides) are all in vitro assays. BL: In picture lower right, in the gel, the lower bands are ejected DNA, and so they are faster. Labelled DNA + Protein complex are slower. As competitor DNA increases, more ejected, so band of ejected DNA becomes stronger. On the right lane, the mutated DNA does not compete for binding, so even at higher concentrations of mutant DNA, it does not eject additional DNA from the protein complex, so stays the same.

Glutathione

Glutathione (GSH) is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides, and heavy metals.[2] It is a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and the amine group of cysteine, and the carboxyl group of cysteine is attached by normal peptide linkage to a glycine. Thiol groups are reducing agents, existing at a concentration around 5 mM in animal cells. Glutathione reduces disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor. In the process, glutathione is converted to its oxidized form, glutathione disulfide (GSSG), also called L-(-)-glutathione. Once oxidized, glutathione can be reduced back by glutathione reductase, using NADPH as an electron donor.[3] The ratio of reduced glutathione to oxidized glutathione within cells is often used as a measure of cellular oxidative stress.

RNA degradation Transcript-specific regulation of decay

Half-lives vary substantially between specific mRNAs (up to 100-fold). Both cis elements and trans factors are involved in this control. 1) Cis elements are usually (but not always) located in 3' UTRs. They can be stabilising or destabilising elements. They are often redundant or work in combination with each other 2) Trans factors can be sequence-specific RNA-binding proteins or micro-RNAs A common destabilising element is called ARE (for AU-rich element): 1) AREs are present in ~8% of mammalian mRNAs 2) If they are added to an mRNA they usually make it more unstable 3) Example: An ARE sequence was added to the 3'UTR of a stable mRNA (β-globin), and the half-lives of mutant (AT) and wild type (GC) mRNAs were compared 4) AREs can be directly recognised by the exosome. However, in most cases they are recognised by RNA-binding proteins that in turn recruit components of the degradation machinery (deadenylases, the exosome, etc)

Glucocorticoid

Has a role in glucose metabolism Synthesized in the adrenal cortex Glucocorticoids are part of the feedback mechanism in the immune system which reduces certain aspects of immune function, such as inflammation. Glucocorticoids affect cells by binding to the glucocorticoid receptor. The activated glucocorticoid receptor-glucocorticoid complex up-regulates the expression of anti-inflammatory proteins in the nucleus (a process known as transactivation) and represses the expression of proinflammatory proteins in the cytosol by preventing the translocation of other transcription factors from the cytosol into the nucleus

Tell me about ampicillin resistance in pBluescript

Has the bla gene The bla gene confers resistance to ampicillin - it encodes β-lactamase, which breaks down the antibiotic.

Prokaryotic Transcription Mechanism High resolution structure of core RNA Polymerase

High resolution structures show how the RNA Pol active site is located at the base of a large cleft formed between the beta and beta prime subunits The cleft is flanked by mobile pincers/jaws, which enclose 20 bp of downstream DNA in elongating complex. The alpha subunits do not form part of the active site. Only the N-terminal domains (NTDs) of alpha are observed in the structure - making nonequivalent contacts with beta and beta prime subunits Alpha subunit C-terminal domain (CTD) is connected to NTD by a flexible linker, and can bind DNA (see later - promoter UP elements and Lac operon) Overall the structure is remarkably similar to eukaryotic RNA polymerase II (see later) despite very low degree of sequence identity and smaller number of subunits. Notably, the regions of sequence identity cluster around the active site.

Processing of pre-mRNA 3' end processing Tell me about A genome-wide view of 3'-end processing

High-throughput sequencing technologies can be used to systematically map cleavage and polyadenylation sites. One strategy is based on the fragmentation of RNA followed by the purification of polyadenylated fragments. These fragments are then sequenced, and the boundary between the gene sequence and the poly(A) tail identifies the cleavage and polyadenylation site. A single experiment can provide this information for hundreds of thousands of individual RNAs. The figure shows an example of a yeast gene that contains three different cleavage and polyadenylation sites, which are used with different efficiencies. Analysis of these data can reveal global trends. For example, more than 50% of human genes are subject to alternative polyadenylation. Polyadenylation site selection is regulated under many conditions. For instance, proliferating cells tend to have shorter 3' UTRs (indicating that proximal cleavage sites are preferred. This has functional importance, as short 3' UTRs have less binding sites for negative regulators of gene expressions (as discussed above for e(r)).

What is ddH₂O?

Highly pure water produced by laboratory purification systems, such as from Milli-Q or similar. dd = double distilled.

What is a His SpinTrap® column?

His SpinTrap® column is suitable for small-scale protein production. It comprises 50 µl of Ni²⁺-Sepharose in a small column that fits into a 1.5 ml microfuge tube and can be centrifuged.

Tell me about the Lon protease family

Hydrolysis of proteins in presence of ATP. ATP-dependent serine protease that mediates the selective degradation of mutant and abnormal proteins as well as certain short-lived regulatory proteins In E. coli, the Lon gene product may proteolytically degrade proteins you incorporated into the E coli, so gotta be careful.

Prokaryotic Transcription Regulation Positive regulation by CAP - 2. RNA Pol recruitment

I think heterologous interactions refers to having a different protein in place of cap, and that protein only recruits RNA Pol. So doesn't matter what protein is there, as long as it recruits RNA pol, thats all it needs to do. UP element just means upstream promoter element

Effect of NaCl, MgCl₂ and urea on melting of DNA....?

IDK YET???? I think urea will denature DNA so melting point will be lowered. DK bout the others yet....

Why does PCR need to have pH 9, alkaline buffer? what happens if you do PCR when pH is acidic?

IDK yet... dna more negatively charged when alkaline..... plays a role? is taq polymerase has very positively active site....? Lower pH and higher temperature both promote depurination... High temperature is necessary for denaturation of DNA, however pH can be controlled. Thats why you go alkaline pH because it reduces depurination of the DNA.

Expression of proteins in E. coli may give rise to fusion proteins Tell me about the proteases used on fusion proteins

IEGR is Isoleucine, glutamic acid, Glycine and Arginine LVPR/GS is Leucine, valine, proline, arginine/glycine and serine

PCR non specific priming?

If primer too short, could bind to other places in the genome. Thus you might amplify a mixture of the sequences. If you are using degenerate primers, the problem may be exacerbated by all the wrong-sequence primers mixed in with the single right-sequence one. Increasing annealing temperature and changing concentration of cations such as magnesium can help improve the specificity of the reaction. Other techniques to improve specificity include: Touch-down PCR Hot start PCR Nested PCR

Tell me about immunofluorescence

Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on microbiological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. The specific region an antibody recognizes on an antigen is called an epitope. There have been efforts in epitope mapping since many antibodies can bind the same epitope and levels of binding between antibodies that recognize the same epitope can vary. Additionally, the binding of the fluorophore to the antibody itself cannot interfere with the immunological specificity of the antibody or the binding capacity of its antigen. Immunofluorescence is a widely used example of immunostaining (using antibodies to stain proteins) and is a specific example of immunohistochemistry (the use of the antibody-antigen relationship in tissues). This technique primarily makes use of fluorophores to visualise the location of the antibodies

What is immunoprecipitation?

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. This process can be used to isolate and concentrate a particular protein from a sample containing many thousands of different proteins.

When you run PCR you form strands with primers (or their complement) at both ends and strands with primers (or their complement) at just one end.

In PCR, you get a geometrically increasing number of strands with primer (or its complement) at both ends, and an arithmetically increasing number of strands with primer at one end. The number of full length DNA molecules remains constant. Brandon:(look at picture. If you start with 1 DNA molecule, there will always be 2 strands of DNA that will have the full sequence original long sequence. These long sequences will generate strands with 1 primer end. So thats the arithmetic bit. But the strands with 1 primer end will generate strands with 2 primer ends. So thats the geometric bit.) The number of strands with primers at both ends becomes the most abundant after just a few rounds of replication. If you run a gel thats mostly what you see. BL: I think the product with 2 primers at both ends is the most likely the smallest one, especially if you are sectioning out only a small portion of the DNA fragment with PCR. So this will be furthest down on the gel.

What is CT value?

In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (ie exceeds background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (ie the lower the Ct level the greater the amount of target nucleic acid in the sample).

Why are DNA probes only labelled on one strand for DNA footprinting and chemical modification interference assays?

In footprinting, you label one end of the strand so that you can pinpoint where the protein binds. If you label both ends of the strand you cannot pinpoint where the protein binds. Due to low concentrations, DNase cleaves the DNA only once, so it splits the DNA into 2 fragments, one fragment will be labelled and seen on the gel, the other will become invisible on the gel. This is very useful because it means that the length of the DNA bands you see on the gel will correspond to how far down the DNA DNAse has cleaved the DNA, so you can pin point where the protein binds. This is the same principle with Modification interference.

Splicing Tell me about Alternative Splicing

In many genes, more than one mRNA is produced by alternative splicing, in which different combinations of splice sites can be paired. Switches in splicing pattern often show tissue specific or developmental control. The availability of EST databases and the advent of ultra-high throughput sequencing have allowed analyses of the prevalence of alternative splicing, by alignment of all sequences derived from the same gene. These studies have revealed that > 90% of human genes are alternatively spliced. In many cases alternative splicing is not and all/nothing phenomenon, that is, several isoforms are made under the same conditions. Alternative splicing can take several forms:

Eukaryotic transcription - activating transcription factors and enhancers Enhancers e.g. Identification of muscle-specific enhancer in the Myosin Light Chain 1/3 gene (MYL1)?

In slide 114 the investigators wanted to locate the cis elements responsible for muscle-specific activation of the MYL1 promoter adjacent to exon 1 (recall that this gene has two widely separated TSSs - UCSC screenshot on slides 96-98). The MYL1 promoter alone was insufficient to activate transcription in myotubes above the basal levels observed in myoblasts. Only after testing numerous genomic fragments in the region of the MYL1 gene did they finally locate a muscle specific enhancer in a 920 bp fragment downstream of the gene, over 25 kB from the MYL1 promoter. Transfections of the CAT reporter plasmids were carried out in a myogenic (muscle) cell line that could either be maintained as undifferentiated proliferating myoblasts (MB) or terminally differentiated into myotubes (MT). The MYL1 enhancer activated expression only in myotubes while the simian virus 40 (SV40) enhancer, acting as a positive control, was active in both MB and MT. Like other enhancers, the MYL1 enhancer consists of a dense array of binding sites for transcription factors spread over ~100 bp. Some of the transcription factors are muscle specific (MYOD1, MYOGENIN, MEF2), while others are more widely expressed.

Why do DNA solutions have a high viscosity?

In the absence of counterions, repulsion between negatively charged phosphate groups keeps the molecule in the extended form, so that DNA in solution behaves as an inflexible rod, hence the high viscosity of DNA solutions.

Prokaryotic Transcription Mechanism Sequence of events during initiation

In the open binary complex, binding of the first NTP is relatively low affinity - can H-bond to template strand, but no stacking interactions. The second NTP binds with slightly higher affinity and allows formation of first phosphodiester bond. Binding of all subsequent NTPs is higher affinity. As successive NTPs are added, the transcription "bubble" expands in a 3' direction (the 5' end is fixed). Multiple cycles of abortive initiation can occur with collapse of the transcription bubble to its original state and release of products of 2-9 nt. Eventually, if the nascent transcript gets extended beyond ~9 nt, the transcription bubble contracts from the 5' end, accompanied by loss of sigma from the complex, marking the successful transition to elongation. The multiple cycles of abortive initiation reflect the fact that there are obstacles in the transition from initiation to elongation. Conceptually, this is perhaps unsurprising given the different requirements of initiation and elongation complexes. The initiation complex needs to be highly sequence specific involving the additional sigma subunit - so that transcription only starts in the right place. On the other hand, the elongation complex needs to be entirely sequence non-specific - it needs to carry on transcribing until a termination signal is encountered. However, it does need to be very stable to ensure processive transcription. Around 15 years ago, high resolution structures illuminated some of the structural transitions that occur during promoter escape...

Tell me about proteins and their interaction with SDS

In the presence of the strong ionic detergent sodium dodecyl sulphate (SDS) CH₃-(CH₂)₁₁-O-SO₃-Na⁺ proteins are completely unfolded. Most proteins bind equal amounts of anionic dodecyl sulfate per gram. (1.4 g per g of protein) and so behave as polyanions with a constant ratio of negative charge to mass. Any inherent differences in charge due to the amino acid side chains are thus obscured and the proteins behave like rigid rods in solution. Provided that disulphide bonds have been reduced (by, for example, b-mercaptoethanol), multimeric proteins are also completely dissociated into their subunits by SDS. During electrophoresis in a polyacrylamide gel that contains SDS, the proteins move at speeds determined only by the size of their SDS-protein complexes (i.e. length of polypeptide chain and therefore molecular mass). For a wide range of protein sizes, the speed of migration falls linearly as the logarithm of the Mr of the protein rises, because longer chains are more retarded by the gel medium

Investigating Mechanisms of Gene Expression In vivo & in vitro assays

In vivo: intact living cells/organism • Physiological conditions • Little control over variables • Difficult to monitor reaction intermediates • Can be unphysiological if test gene or expressed RNA too abundant In vitro: test in a cell extract • Precise control over reaction conditions • Can purify trans-factors (protein or RNA) • Often inefficient • Unphysiological - not all components present at appropriate levels In practice: correlate behaviour of endogenous gene with test gene in vivo & in vitro.

What are inclusion bodies?

Inclusion bodies, sometimes called elementary bodies, are nuclear or cytoplasmic aggregates of stable substances, usually proteins. They typically represent sites of viral multiplication in a bacterium or a eukaryotic cell and usually consist of viral capsid proteins. Inclusion bodies can also be hallmarks of genetic diseases, as in the case of neuronal inclusion bodies in disorders like frontotemporal dementia and Parkinson's disease

Selenomethionine labelling?

Incorporation of selenomethionine into proteins in place of methionine aids the structure elucidation of proteins by X-ray crystallography using single- or multi-wavelength anomalous diffraction (SAD or MAD). The incorporation of heavy atoms such as selenium helps solve the phase problem in X-ray crystallography (Using the ability of certain elements to absorb X-rays and change the diffraction intensities). Seleno-Methionine can be incorporated in the place of methionine during protein expression (typically in E.coli).

How to transform E coli?

Incubate with CaCl₂ solution on ice followed by a heat shock. More efficient uptake of DNA can be achieved by electroporation, treatment with a high voltage electric shock.

Splicing Regulation of splicing by RNA-binding proteins Splicing repressors:

Inhibit the use of splice sites. Example: hnRNPs (heterogeneous nuclear ribonucleoproteins) a) Modular structure: contain an RNA-binding domain (one or two RRMs or other RNArecognition motifs) and a protein interaction domain (sometimes the RRM motifs themselves) b) hnRNPs repress splicing by several mechanisms. For example, the PTB(an example of an hnRNP I think) protein inhibits some splice sites by preventing the binding of U2AF BL: I think the picture represents an hnRNP gene, so in the protein there is a glycine rich part, so in PTB maybe the back part has a lot of glycines?

Splicing Tell me about its discovery

Introns were discovered in 1977, and Philip Sharp and Richard Roberts received the Nobel Prize in 1993 for their 'discovery of split genes'. They used adenovirus, a virus that infects mammalian cells and produces mRNAs that are capped, spliced and polyadenylated. One of the key experiments was the analysis of the so-called R loops between mRNA and DNA: 1) mRNA is hybridized to the corresponding doublestranded DNA fragment under conditions that favor DNA-RNA interactions over DNA-DNA annealing 2) mRNA anneals to the DNA and displaces one strand of DNA, leading to the formation of a loop of single stranded DNA (R loop) 3) The nucleic acids are visualized by electron microscopy. Single- and double- stranded nucleic acids can be distinguished by their width 4) When the technique was applied to adenovirus DNA, it was found that the mRNA had 'tails' protruding on both ends (a 3' end tail could be explained by poly(A), but a 5' tail could not) 5) If the R loops are incubated with a DNA fragment from a separate (non-contiguous) part of the virus genome they anneal to it, showing that the mRNA is a composite molecule made up from fragments complementary to separate regions

Tell me about oligo (dT) cellulose chromatography

Isolates only those RNAs with a poly(A) tail.

Tell me about ribosome binding site

It allows us to direct translation initiation. In E. coli, this is done by a ribosome binding site. This is composed of a sequence of a few nucleotides in the mRNA, typically AGGAGG (also called the shine-dalgarno sequence) shortly before the translation initiation codon. It is complementary to a few nucleotides at the 3' end of the E. coli 16S rRNA. Translation starts at the first AUG codon in the mRNA downstream from the ribosome binding site.

When identifying important residues and domains - what do you know about analysing the function of individual protein domains...

It is often useful to look at the function of individual domains within a protein. • PCR may be used to amplify the sequence encoding each protein domain and clone it into a suitable plasmid - E.g. for expression in mammalian cells to look at a particular function. This is only possible if individual domains are encoded by contiguous sequences. • It is important to ensure that the domain boundaries are appropriate - if the cloned sequence encodes an incomplete domain, it may not fold properly or be soluble if trying to make recombinant protein. Information regarding domain boundaries can come from analyzing homologous proteins, from structural studies, domain prediction programmes etc. • It's worth bearing in mind that individual domains may not behave as they would in the context of the whole protein.

What do we do once we have a clone? Identifying important residues and domains

It is often useful to make specific mutations in DNA sequences in order to investigate the importance of particular amino acids - E.g. for structural studies, enzymology, nucleic acid binding etc. In addition, mutagenesis may be used to analyze the importance of putative regulatory sequences in DNA or RNA. In addition to investigating the function of specific amino acids, the relevance of whole protein domains may be analyzed to determine their functional significance. 1) Mutagenesis 2) Analyzing the function of individual protein domains Brandon: I think first you must do mutagenesis and make proteins with the functional domain and without THEN you analyze the protein domains.

analyzing the effect of reducing the expression level of a protein... Tell me about Gene Disruption using cre lox

It is possible to knockout a gene by gene disruption - knocking out endogenous genes by swapping them by homologous recombination with a mutant one. Controlled excision It is also possible to get controlled excision of genes. This relies on a site-specific recombination system from a bacteriophage, the cre-lox system. The phage Cre protein is a recombinase that directs recombination at a specific sequence, lox. This can be exploited as follows: 1) Replace target gene by a copy flanked by lox sequences 2) Supply Cre protein (E.g. by inserting a cre gene under a controllable promoter, and then inducing expression). It is possible to express Cre in specific tissues, so can get selective gene inactivation in those tissues. This is particularly useful if inactivation of a gene throughout an organism is lethal. 3) Sit back and wait. Cre mediates recombination across lox, causing excision of the gene. This is sometimes called "floxing", as it removes sequences that are flanked by lox elements. This can be used to make conditional knockout mice... for example, where the Cre-lox system is used to get tissue specific gene disruption.

Analyzing the localization of proteins?

It is possible to make use of fusion proteins to analyze the localization of a protein - by tagging a protein in a way that makes it easy to identify. Tagged proteins may be visualized directly (E.g. GFP), or may provide a means of easily recognizing a protein via interaction with specific antibodies (E.g. Epitope tags - Flag, HA, Myc tags). 1) GFP-tagged proteins 2) Epitope-tagged proteins 3) Reporter genes

What is the hydrophobic effect?

It is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water molecules. Picture: A droplet of water forms a spherical shape, minimizing contact with the hydrophobic leaf.

What is TG1 E coli?

Its a strain of E coli. Not much on google about it.

Tell me about Taq polymerase

Its from Thermus aquaticus, a thermophilic BACTERIUM. 94 kDa protein. 1) Optimum Temp for DNA synthesis is 72-75 C 2) 5'-3' DNA pol incorporates 50-60 nt/sec at 72 C 3) Has 5'-3' exonuclease 4) has NO 3'-5' proofreading activity. 1 in 10⁴ nucleotides are incorrect 5) Half life at 95 C of 40 minutes. 6) Taq polymerase has relatively low processivity. This means it is likely to dissociate from the template before it has synthesized a long piece of DNA. Sometimes, dissociation is caused by the incorporation of an incorrect nucleotide. The polymerase cannot correct the error, but it cannot readily elongate the strand being synthesized, as the 3' end is not base-paired to the template. So it dissociates. The low processivity places a maximum size limit of molecules that can be amplified using this enzyme, which is typically 2-4kbp. Brandon: tends to dissociate when incorporates wrong base pair.... this means that target DNA lengths will be more accurate than I thought.... When you do PCR, you run the DNA synthesis step for a minute or so, so likely to be significant loss of activity after doing a full set of cycles.

Kanamycin

Kanamycin A, often referred to simply as kanamycin, is an antibiotic used to treat severe bacterial infections and tuberculosis. Kanamycin interacts with the 30S subunit of prokaryotic ribosomes. It gives rise to substantial amounts of mistranslation and indirectly inhibits translocation during protein synthesis.

How would you digest the PCR product using restriction enzymes?

Key to buffers: • CutSmart™ (supplied as 10x). 1x Buffer: 50 mM Potassium acetate, 20 mM Trisacetate, 10 mM Magnesium acetate, 100 μg/ml BSA Add the following to a 1.5 ml microfuge tube: 15 µl purified PCR product (from above) 5 µl of 10x restriction enzyme buffer (CutSmart™) 28 µl H₂O Total 48 µl A demonstrator will add 1 µl BamHI and 1 µl HindIII to the tube. Mix gently and briefly spin in the benchtop microcentrifuge using an empty tube as a balance. Incubate the tube in the 37 °C heat block for 30 - 45 min

What are ligand binding assays?

Ligand binding assays (LBA) is an assay, or an analytic procedure, whose procedure or method relies on the binding of ligand molecules to receptors, antibodies or other macromolecules

Tell me about propeller twist?

Literally imagine a propeller twisting. It is twisting in an axis perpendicular to the axis of the helix.

Tell me about enhancer trapping

Makes gene mutagenesis and initial cloning of genes relatively easy. Basis of enhancer trap techniques: Insertion of P-transposable element. The P-elements use in enhancer trap analysis have had the transposase gene completely removed, which means that once the P-element is inserted it is stable and cannot jump around. P-elements are engineered to also have lacZ reporter gene. You often do enhancer trapping with drosophila, so the P-element was initially inserted by injection into embryos at the syncytial blastoderm stage when nuclei are dividing but cellularization has not yet occurred. However, transposase gene must also be added to transposase can be produced, but the transposase gene was modified so it cannot be incorporated into the genome (has had inverted repeats removed) The initial insertion of the P-element into the genome may result in the mutation of a gene. In many cases, however, the initial insertion of the P-element does not cause an observable mutant phenotype. This usually occurs because the P-element has been insered into non-coding and non-regulatory regions of DNA. The second useful feature of the P-element is the lacZ gene. It is the presence of this gene that gives these techniques their name. The presence of this gene in the P-element allows us to know where and when genes affected by the P-element insertion are expressed. The lacZ gene codes for ß- galactosidase, an enzyme that is produced in E. coli but not Drosophila. ß-galactosidase, in the presence of the correct substrate and the indicator chemical, X-gal, produces a blue color in tissues where it is expressed. The lacZ gene on the P-element is expressed only weakly unless it is activated by enhancers in the vicinity of the P-element. Nearby enhancers that normally activate Drosophila genes in the vicinity of the P-element insertion will also activate the lacZ gene on the P-element. The lacZ gene will be expressed with the same tissue specificity and temporal pattern as the nearby Drosophila gene that normally is regulated by these enhancers. Therefore, tissues that express the Drosophila genes driven by these enhancer elements will turn blue in the presence of X-gal. The Drosophila enhancer has in effect been trapped into telling us something about the tissue specificity and temporal expression pattern of genes in the vicinity of the P-element insertion. The genes driven by these enhancer elements are also likely to be the genes mutated by the insertion or remobilization of the P-element. This reporter gene system is not always accurate. Sometimes a nearby gene with a strong enhancer will drive the lac Z expression while the insertion of the P-element causes the disruption of a different adjacent gene. The power of enhancer trap techniques is that the simple insertion of a P-element into the genome of a fruitfly creates mutants and allows us to obtain genomic clones in the area of the mutation, to map the mutated gene to a physical location on the chromosome and to know the tissue specificity and expression pattern of the gene of interest.

A: Fusion with MalE maltose binding protein (MBP) The resulting protein can be purified on an Amylose column - and eluted with maltose (competes for binding). Why?

Maltose is a dimer of glucose with an alpha 1-4 glycosidic linkage. Amylose is a polymer linked in the same way.

Regulation of translational efficiency in bacteria Translational coupling

Many bacterial mRNAs are polycistronic. Each cistron has its own SD sequence, so ribosomes can access each initiation site independently. In principle, a ribosome initiating at a downstream cistron should not be dependent on translation of the upstream cistron. In some cases, however, this is the case. This phenomenon is known as translational coupling. e.g. MS2 phage: a) A nonsense mutation in codon 6 of the coat protein gene blocks translation of the downstream replicase gene (this mutation is said to be 'polar' as upstream genes are unaffected) b) By contrast, nonsense mutations at codons 50-70 of coat protein gene do not affect replicase expression. c) Explanation: the secondary structure around the SD of replicase gene has to be unwound by ribosomes translating the coat protein gene to be accessible

RNA localization

Many mRNAs are localised to specific subcellular compartments, leading to the asymmetric localisation of the proteins they encode. 1) RNA localisation is essential for embryogenesis (embryonic determinants), neuronal function (by allowing independent regulation of protein expression in axons/dendrites) and yeast differentiation (asymmetric division) 2) It is regulated by cis signals (mostly in UTRs) and trans factors (RNA-binding proteins) 3) It is often coupled with translational repression of mRNAs before localisation We will study the ASH1 mRNA as a paradigm of the mechanisms involved in the asymmetric localisation of mRNAs. The yeast Saccharomyces cerevisiae reproduces by forming a bud that will eventually form the daughter cell. The ASH1 mRNA is asymmetrically localised at the tip of the bud. This leads to the specific production of the ASH1 protein in the daughter cell. The ASH1 protein functions as a transcription factor that represses transcription of its targets ASH1 mRNA localisation is controlled by cis-sequences in the 3' UTR. Evidence: 1) Make reporter containing ASH1 or a control (ADH1) 3' UTR 2) Follow RNA localisation by FISH (fluorescence in situ hybridisation): Cells are fixed, incubated with a fluorescently labelled probe complementary to the reporter sequence and visualised using fluorescence microscopy 3) The RNA containing the ASH1 3' UTR is localised to the bud ASH1 mRNA is transported to the bud by the actin cytoskeleton. Mechanism: 1) An RNA-binding protein (SHE2) recognises cis sequences in the 3' UTR of ASH1 2) An adaptor protein (SHE3) connects SHE2 to a myosin (actin-dependent molecular motor) 3) Myosin transports the complex along actin filaments to the bud

Methods to study posttranscriptional processes Tell me about Mapping RNA-protein interactions

Many methods to study the interactions between protein and RNAs are very similar to those used for DNA-binding proteins. Some of them are are based on the purification of specific RNAs or proteins (affinity methods) RNA purification (e.g. RNA tagging using biotin) o An RNA oligonucleotide is synthesized in vitro using biotinylated UTP o The biotinylated oligonucleotide is incubated with purified proteins or cell extracts o The RNA-protein complexes are recovered using streptavidin beads (streptavidin binds to biotin with very high affinity) o Bound proteins are detected by Western blot or other methods Protein purification o Analogous to ChIP (see Prof Luisi's lectures) o A protein is purified together with associated RNAs using specific antibodies o cDNAs are generated from the mRNAs using reverse transcriptase o Regions of interest are detected by PCR o Alternatively, the cDNAs can be identified using DNA microarrays or sequencing UV cross-linking o The identification of many RNA-protein interactions requires that the associations be stabilized by cross-linking o RNA-protein complexes are irradiated with UV light (can be done in vivo or in vitro) o UV induces formation of stable covalent bonds between RNA and proteins

In RNA, there are 28 possible base-pairs with two or more H-bonds?

Many of the 28 base-pairing interactions with 2 or more H-bonds have been observed in structures of rRNAs, tRNAs, riboswitches, spliceosomes, ribozymes and other folded RNAs Only the Watson-Crick base pairs are suitable for information storage in DNA; these complementary base-pairs are isomorphic and can adopt the regular doublehelical structure. The other base-pairing interactions allow RNA (or, in principle, single stranded DNA) to adopt a multitude of conformations. So in an essay you would mention this fact to show why RNA can adopt so many different conformations, and perhaps why it can have enzymatic activity, shape can be so varied. e.g. the G-G N1-carbonyl N7 amino base-pair underlies four-stranded Gquadruplex structures. Each G makes four H-bonds. O6 and N7 on the Hoogsteen face are H-bond acceptors, while N1 and exocyclic N2 on the Watson-Crick face are H-bond donors. Examples of 28 possible base pairs: Watson crick, reverse watson crick, A-U Hoogsteen, A-U Reverse Hoogsteen, GU wobble, GU reverse wobble, A-A pairing, etc

What is Metallothionein?

Metallothionein (MT) is a family of cysteine-rich, low molecular weight (MW ranging from 500 to 14000 Da) proteins. They are localized to the membrane of the Golgi apparatus. MTs have the capacity to bind both physiological (such as zinc, copper, selenium) and xenobiotic (such as cadmium, mercury, silver, arsenic) heavy metals through the thiol group of its cysteine residues, which represent nearly 30% of its constituent amino acid residues. Brandon: so basically, using its cysteine residues it can soak up loads of heavy metals. Why is it localised to the Golgi membrane? Maybe when it soaks up enough heavy metals, it goes into a vacuole to sequester itself from the cell.

use of cultured embryonic stem (ES) cells when making transgenic animals to analyze the function of your protein... Tell me about using the Cre System

Method: • The ES cells are used to introduce DNA constructs that give rise to homologous recombination with our target gene, based on the method described above. Various positive selection markers (E.g. neomycin) are used that enable selection of cell lines carrying the mutation of interest. Negative selection markers are sometimes also used (E.g. Thymidine kinase) to ensure proper recombination (they are eliminated if the recombination occurs correctly). • The individual transformants are cultured and then analyzed to ensure that the inserted DNA has integrated by homologous recombination. E.g. Using PCR. • Having identified a cell line in which the DNA is integrated by homologous recombination, cells are injected into developing embryos to make transgenic animals. Mosaic animals will typically result. • Mosaic individuals with can then be mated to non-transgenics, to form transgenic progeny. A progeny individual that derived from a transgenic germ cell will be fully transgenic, not mosaic (it will be heterozygous, though). So injecting modified ES cells allows us to regenerate mosaic individuals. They can then be crossed (see above) to make non-mosaic individuals, with one copy of the gene knocked out. In other words, they are heterozygous - crossing can be used to generate homozygotes. These are often called "knockout mice" (or heterozygous knockouts). Generation of homozygotes are not always possible is the gene is essential for life. Tissue-specific knockouts can also be generated using the Cre system.

Tell me about the Method itself of Gibson assembly method

Method: • Design primers to PCR amplify fragment(s) (and/or vector) with appropriate overlaps (E.g. 40 bp) - high fidelity polymerase o Computer programmes exist to automate much of the process of designing constructs for Gibson Assembly - E.g. Gibthon Construct Designer (http://www.gibthon.org) • Combine DNAs in Gibson Assembly Reaction - and for each pair of overlapping fragments: • T5 exonuclease creates single-stranded 3' overhangs that facilitates the annealing of fragments with complementarity at one end (overlap region) • Polymerase fills in gaps within each annealed fragment (5' to 3') • DNA ligase seals nicks in assembled DNA So using this protocol you efficiently generate fully assembled DNA.

Difference between cloning and subcloning?

More specifically, cloning describes the procedure by which an original (primary) cDNA or gene is obtained, e.g. from a library. Subcloning describe the subsequent manipulations of that clone, change of vector, subcloning fragments, etc. the use of the word "subcloning" also means to split a large fragment to be cloned into two or more pieces (i.e. subclones) to be then reassembled in your final destination/expression vector. This is generally done when the fragment to clone is to large to be cloned at once, so one creates building blocks/pieces or subclones that will come together in your final vector.

Tell me about neomycin

Neomycin belongs to aminoglycoside class of antibiotics that contain two or more aminosugars connected by glycosidic bonds. Neomycin binds to the 30S subunit of the ribosome and inhibits translation of proteins from mRNA.

Tell me about Oct-1

Oct-1 is a ubiquitous transcription factor in mammalian cells that regulates transcription of some common genes. Oct-1 can also regulate the transcription of cell-specific genes, e.g. immunoglobulin genes in B cells, by recruiting co-activators Oct-1 is known to bind to DNA via a small domain of 162 amino acids called the POU domain.

What is OmpT

OmpT is an aspartyl protease found on the outer membrane of Escherichia coli. OmpT is a subtype of the family of omptin proteases, which are found on some gram-negative species of bacteria

What are the 7 things we want to do once we have a clone?

Once we have a clone, there are various things we may want to do. If the clone encodes our favourite protein, it is likely that we may want to do experiments to study the function of the gene. We will therefore consider a number of experimental questions you may want to address, and look at some key techniques that may be used to carry out the relevant experiments. 7.1 Analyze Gene Expression 7.2 Performing in vitro assays 7.3 Analyzing the localization of proteins 7.4 Identifying interacting proteins 7.5 Identifying important residues and domains 7.6 Analyzing the effect of reducing the expression level of a protein 7.7 Making transgenic animals to analyze the function of your protein

What is Hot start PCR?

One cause of non-specific priming is that before the reaction mix has reached the right temperature for denaturation, priming and thus DNA synthesis may already have started. (primer binds to wrong place when temperature is still too low) In hot start PCR the polymerase is not active until the denaturation temperature has been reached. Can be done simply by adding the polymerase later yourself, or using a polymerase that is only activated at the initial denaturation step. E.g. GoTaq Hot Start Polymerase (Promega) - this polymerase is kept inactive by having an antibody bound at temperatures <70⁰C

Processing of pre-mRNA 5' end processing: capping Tell me about capping specificity

Only di- or tri-phosphate ends are capped (mRNAs digested by endonucleases are not capped) All Pol II transcripts are capped Only Pol II transcripts are capped. Conversion of a Pol II promoter to Pol I or III results in no capping, even though transcript sequence is the same

Yeast 2 Hybrid System

Page 31 of Gene cloning and manipulation BMB Dr Dee Scadden In this instance, you are using S. cerevisiae to screen a library of genes encoding unknown proteins for proteins that interact with a protein whose gene you've already cloned. The most common version makes use of the S. cerevisiae transcription factor GAL4. GAL4 has 2 domains - a DNA binding domain and an activation domain. When the 2 are joined together (as they usually are) they can activate transcription of a gene downstream of the DNA binding site. But they don't have to be joined directly - they can be connected by pairs of interacting proteins. By exploiting property of GAL4, you can look for interacting proteins. A hybrid is made encoding a fusion between the DNA binding domain from GAL4 and the protein you've already cloned (the "bait"). This is cloned into a suitable plasmid - E.g. a yeast episomal plasmid (YEp) based on the 2 μm endogenous plasmid. The library you're screening is cloned into a suitable vector generating a fusion between the activation domain and the library members. Many different fusion proteins are thus produced - all of which contain the activation domain fused to a random protein. The library is introduced into a host that: • Contains the first construct (bait) and • The DNA binding site upstream of a selectable reporter gene (E.g. HIS3). When a cell gets a library construct encoding a protein that can interact with the bait protein, they effectively glue the activator and DNA-binding domains of GAL4 together, and transcription is subsequently activated. In the case of the HIS3 reporter gene, that results in HIS3 transcription, the ability to synthesize histidine and thus the ability to grow in the absence of added histidine: So the members of the library that confer growth on the selective medium are likely to encode something that interacts. This system is very useful, but can be prone to false positives or negatives.

Why is size of the product a difficulty for PCR?

PCR is most efficient at amplifying smallish stretches of DNA (up to 2-3kb), the amplification efficiency (and therefore the yield of amplified fragments) decreases significantly as amplicon size increases over 5 kb. This decrease in yield can be attributed to the accumulation of truncated products, which are not suitable substrates for additional cycles of amplification. Truncated products accumulate because Taq polymerase has a low processivity These products appear as smeared, as opposed to discrete, bands on a gel. It is however possible to amplify larger pieces of DNA - often using a mixture of polymerases. Longer extension times can increase the yield of longer PCR products because fewer partial products are synthesized. In addition, template quality is crucial. Depurination of the template, which is promoted at elevated temperatures and lower pH, will result in more partial products and decreased overall yield. In long PCR, denaturation time is reduced to 2-10 seconds to decrease depurination of the template. Additives such as glycerol and dimethyl sulfoxide(DMSO), also help lower the strand-separation and primer annealing temperatures, alleviating some of the depurination effects of high temperatures.

Tell me about Blunt cloning

PCR often generates a product with blunt ends, which can be cloned into a vector linearised with an enzyme that also gives blunt ends (E.g. EcoRV, SmaI). Blunt cloning is not as efficient as cloning using 'sticky ends', but it is still possible. A disadvantage of blunt cloning is that the insert can go into the vector in either orientation - 'non-directional' cloning

What is POU?

POU is a DNA binding domain from a transcription factor called Oct-1 The POU domain itself is composed of two subdomains: the homeodomain (PouH) and the POU specific domain (PouS). Homeodomains use a helix-turn-helix motif to bind DNA and are found in several other proteins. The POU specific domain, as its name suggests, is only found in POU domain proteins. The structure of the complex of the Oct-1 POU domain with DNA has been solved by X-ray crystallography and it shows that both the subdomains bind to DNA.

Tell me about using mutagenesis in identifying important residues and domains

Page 33 Scadden Use of oligonucleotides to introduce mutations is very standard, especially for the generation of point mutations or small insertions/deletions. There are lots of variations on a theme. In one basic approach we anneal a chemically synthesized 'mutagenic' oligonucleotide containing the mutation required to a cloned copy of the target gene (there will therefore be a mismatch of a base pair or so). A second strand is completed, and the molecule transformed back into E. coli. Host DNA repair and replication will do (or undo) the rest. It is possible to bias the repair process in favour of the mutant strand - helps makes the mutagenesis more efficient. Oligonucleotide mutagenesis can also be done very conveniently using PCR. You just make a pair of primers corresponding to the mutated sequence, rather than the original, and amplify a gene fragment or the whole plasmid containing the sequence to be mutated. However, it is necessary to do the PCR with a high-fidelity polymerase to minimise the potential for errors (E.g. Pfu, Pwo Phusion DNA polymerase etc.). The amplified sequences should be sequenced following mutagenesis to ensure that no errors have been introduced. Primer design: • Both primers must contain the desired mutation(s) and anneal to the same sequence on opposite strands of the plasmid • Primers should be 25-45 nt in length, with a melting temperature (Tm) of ≥78 °C. Using primers longer than 45 bases increases the likelihood of secondary structure formation, which may reduce the efficiency of mutagenesis • The desired mutation should be in the middle of the primer with 10-15 bases of correct sequence on both sides (particularly for multiple mutations) • The primers should have a minimum GC content of 40% and should terminate in one or more C or G bases • Primers should be purified before use to optimize mutation efficiency Digestion with DpnI: DpnI specifically digests methylated or hemi-methylated DNA • DNA isolated from the majority of E. coli strains is dam methylated and therefore susceptible to digestion by DpnI So - the parental DNA strands are specifically digested by DpnI, leaving only the newly synthesized mutant DNA Because PCR can introduce mutations itself, the products always need to be sequenced following mutagenesis to make sure secondary mutations haven't been included.

F factor in E. coli?

Plasmids are usually independent of the main chromosome. The F factor is the exception, it can become integrated into the main chromosome.

Tell me about the pET system

Powerful system developed for cloning and expression of recombinant proteins in E. coli. . Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription. ; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that almost all of the cell's resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction

Tell me about preparing in vitro transcribed RNA

Preparing in vitro transcribed RNA (pp162-164) Plasmids are available that contain phage promoters that are recognised by specific phage RNA polymerases (E.g. SP6, T7, or T3), so can be used to synthesise RNA in vitro. For instance, pBluescript contains T7 and T3 RNA polymerase promoters. Phages normally direct the synthesis of their own RNA polymerases on cell infection, which are highly specific for phage promoters. For in vitro RNA transcription, the following steps are performed: • The DNA sequence is cloned into the vector in a site just downstream from one of the powerful phage promoters (the MCS is contained between the two phage promoters). The orientation of the insert and choice of RNA polymerase will determine which strand of the cloned sequence is transcribed. • The plasmid is linearised at the end of the coding region to be transcribed (to prevent circular transcription). • The construct is then incubated with phage polymerase in an in vitro reaction (with a suitable buffer, NTPs, Cap analogue etc.). Because the polymerase is so specific for phage promoters, a pure transcript of the region downstream from the phage promoter is produced. Other sequences don't get transcribed. The transcript may need a 5' cap (see last year's Cells lectures). This can be done in vitro after transcription, or more conveniently by including a Cap analogue (GpppG) in the transcription reaction as well as the more usual pppG (i.e. GTP). The GpppG can be incorporated at the beginning of the transcript (which usually begins with G), but not elsewhere.

Processing of pre-mRNA

Processing of pre-mRNA Eukaryotic mRNAs are processed by the addition of a cap at the 5' end, a poly(A) tail at the 3' end and the removal of introns. These processes take place in the nucleus, and they are often cotranscriptional (i.e. occur while transcription is still taking place). 1) 5' end processing: capping 2) 3' end processing - Most eukaryotic mRNAs are modified by the addition of a poly(A) tail at their 3' end. The tail is added in a two-step mechanism, as discussed in another card. 3) Splicing

Tell me about BL21(DE3)

Protein Expression... The Oct-1 POU domain will be expressed in an E. coli strain called BL21(DE3). BL21 is often used for protein expression because it is deficient in two proteases (Lon and OmpT) that might otherwise degrade the recombinant protein, in this case to POU domain. the (DE3) part of the name means that there is a DNA insert in the bacterial chromosome, which encodes the T7 polymerase under the control of a highly efficient promoter derived from the lac promoter so we can switch on its expression with IPTG, the lactose analogue

Tell me about T7 promoter

Protein Expression... This T7 promoter is used as it is small, the T7 RNA polymerase (T7 promoter is recognised by RNA polymerase from bacteriophage T7) binds to and initiates within a 17 bp sequence, and has a high rate of initiation and processivity so it makes a lot of mRNA.

Tell me about T7 RNA polymerase

Protein Expression.... T7 RNA polymerase is not normally present in E. coli and the (DE3) part of the name means that there is a DNA insert in the bacterial chromosome, which encodes the T7 polymerase under the control of a highly efficient promoter derived from the lac promoter so we can switch on its expression with IPTG, the lactose analogue.

Tell me about PKR

Protein kinase R (PKR) is activated by dsRNA and is part of the antiviral response (it shuts down protein synthesis in infected cells) Protein kinase R (PKR) is activated by dsRNA and is part of the antiviral response (it shuts down protein synthesis in infected cells)

Tell me about RNA recognition motifs

RNA recognition motif, RNP-1 is a putative RNA-binding domain of about 90 amino acids that are known to bind single-stranded RNAs. It was found in many eukaryotic proteins RRM proteins have a variety of RNA binding preferences and functions, and include heterogeneous nuclear ribonucleoproteins (hnRNPs), proteins implicated in regulation of alternative splicing (SR, U2AF2, Sxl), protein components of small nuclear ribonucleoproteins (U1 and U2 snRNPs), and proteins that regulate RNA stability and translation (PABP, La, Hu).[2][3][5] The RRM in heterodimeric splicing factor U2 snRNP auxiliary factor appears to have two RRM-like domains with specialised features for protein recognition.[6] The motif also appears in a few single stranded DNA binding proteins. The typical RRM consists of four anti-parallel beta-strands and two alpha-helices arranged in a beta-alpha-beta-beta-alpha-beta fold with side chains that stack with RNA bases. A third helix is present during RNA binding in some cases

Tell me about RNA interference - post transcriptional gene silencing, in analyzing the effect of reducing the expression level of a protein

RNAi is a powerful method for specifically reducing gene expression in various cells or organisms. It is used experimentally in a wide range of organisms, from Drosophila and Caenorhabditis upwards, including plants. RNAi is often used as an experimental tool for knocking down your favourite protein in your chosen system. • In organisms such as Drosophila or C. elegans, long dsRNA is often used to initiate RNAi (dsRNA is either introduced directly, or synthesised within the organism - E.g. C. elegans could be fed E. coli expressing dsRNA). • In mammalian cells, use of long dsRNA is not possible as it triggers an interferon response and cell death. Instead, siRNAs may be used to initiate RNAi. siRNAs can be used to transfect mammalian cells directly using Lipofectamine®-2000 (Life Technologies) (i.e. A cationic liposome formulation, which complexes with negatively charged nucleic acid molecules - allows them to overcome the electrostatic repulsion of the cell membrane. DNA-containing liposomes (with positive charges on their surfaces) can then fuse with the negatively charged plasma membrane of living cells, allowing the nucleic acid to cross into the cytoplasm). • Alternatively, plasmids that express short hairpin RNAs (shRNAs) can be used to transfect mammalian cells - the shRNAs are then processed in the cell like miRNAs to give rise to the guide strand that will subsequently target the mRNA. • shRNAs can be transiently or stably expressed in mammalian cells. o E.g. stable cell lines may be produced using Flp-In™ 293 T-Rex cells - in this instance you get targeted integration of the sequences to produce shRNAs at a specific site (FRT ) Flippase Recognition Target) site - use Flp recombinase to get integration) - so you know that it is inserted at a transcriptionally active genomic locus that will give high level expression. • Expression of shRNAs could be constitutive or inducible (E.g. using tetracycline) • Western blots are performed to determine whether your protein has been knocked down by RNAi - and to what extent. If a suitable antibody is not available, you can alternatively look at mRNA levels using RT/qPCR. However, while this will tell you if you have knock down at the RNA level, it will not tell you if the protein level is reduced - and if your protein has a long half-life, it may persist in the cell despite the mRNA level being reduced. It may then be difficult to draw conclusions as to the function of your protein if you are not sure whether it is still present in the cell.

RNA degradation

RNAs are degraded in the cell by specific enzymes called ribonucleases. Ribonucleases that degrade RNAs from the ends are called exonucleases (they can do it in the 5'->3' or in the 3'->5' direction), while those that cut RNAs in the middle are called endonucleases. RNA degradation is carried out by several multiprotein complexes. It is a transcript-specific and highly-regulated process that is controlled by both cis-elements and trans-acting factors. In these lectures we will only discuss eukaryotic RNA degradation, although prokaryotic mRNAs are also degraded specifically. The process of RNA degradation is also called RNA turnover or RNA decay.

Ribosome structure and function Termination of translation: a structural view

Release factors mimic the shape of a tRNA molecule. One domain of the protein is localized close to the PTS and induces the hydrolysis of the peptidyl-tRNA, while another one directly recognizes the termination codon on the mRNA. RF1 and RF2 recognise specifically termination codons on mRNA: a) Mutational analysis revealed that the specificity is conferred by a single tripeptide -> called the peptide 'anticodon' b) 3d structures of RF1 bound to the ribosome confirm the model

Reporter genes can also be used to analyze the effects of other regulatory elements....?

Reporter genes can also be used to analyze the effects of other regulatory elements - E.g. the effects of miRNA binding sites in the 3' UTR could be analyzed using reporter genes. • miRNAs regulate gene expression by binding to specific sequences in the 3'UTR of specific mRNAs • miRNA binding results in translational repression and mRNA decay • Analyze efficiency of repression using reporter genes - use a reporter that is easy to assay - E.g. Luciferase Brandon: in picture, RL-con probably stands for RL control, no sites. So you would have 3x miRNA binding sites and a control. But the thing is, when miRNA binds to RL, which is the reporter, less miRNA available to bind to the original target.... so gotta be careful....

Regulation of translational efficiency in bacteria Regulation by riboswitches

Riboswitches are RNA domains within a sequence that control gene expression in response to small molecules 1) The presence/absence of the ligand changes the riboswitch secondary structure (between mutually exclusive conformations) and controls gene expression 2) Usually act in cis 3) Contain two domains: aptamer (binds to the ligand) and expression platform (interacts with the transcriptional or translational machinery) Riboswitches regulate gene expression by attenuation (c) or translational control (d) Guanine-binding riboswitch is an example to illustrate how riboswitches can detect specific ligands (another card)

What factors should you think about when doing primer design?

SMMLD 1) Length - too short then by chance bind somewhere else. If too long might form secondary internal structures. 2) Mismatches 3' end of primer should be correctly base paired to the template, otherwise polymerase will not be able to extend it. Have C or G as the terminal nucleotide at the 3' terminal. You could put some mismatches in if you want to a lower the temperature at which primer dissociates(advantages?... probs not?) 3) Melting temperature - the temperatures at which the two primers can associate with the template should be relatively similar to ensure that they both bind at about the same time as temperatures are being lowered during annealing. Similarity of temperatures means primers should have a similar nucleotide composition. 4) internal secondary structure - this should be avoided, or a primer may fold back on itself and not be available to bind to the template. As an intra-molecular reaction, self-annealing is likely to take place in preference to intermolecular annealing of the primer template. 5) Primer-primer annealing. It is important to avoid the two primers being able to anneal to each other. Extension by DNA polymerase of two self-annealed primers leads to formation of a primer dimer. These will be very efficient templates for amplification in subsequent rounds of PCR, as they are small.... so these will replicate instead of the DNA you want so avoid primer-primer annealing.

Chromatin Packaging DNase hypersensitive sites - nucleosome free regions

Sensitivity of DNA in chromatin to digestion by DNase I was an early form of biochemical evidence indicating a change in chromatin packaging in expressed genes compared to non-expressed genes. The first experiment showed that expressed genes are digested by DNase I about ~5-fold more rapidly than bulk chromatin. This extends across the length of the gene. However, they are still occupied by nucleosomes (see e.g. slide 80), so the enhanced DNase sensitivity may reflect chromatin de-condensation, perhaps by a 30 nm to 10 nm fibre transition (see following slides) The DNase I hypersensitivity assay provides a higher resolution view showing specific locations that are digested by DNase I, 10-20 fold faster than the rest of the gene. These really are nucleosome-free regions, due to binding of transcription factors at promoters or enhancers. The genes are sometimes "poised" to be activated rather than continually active. The poised state, with DNase hypersensitive sites could be established at the previous round of DNA replication. NB: nucleosome-free regions were not seen in slide 80, because the plot shows the average density of MNase resistant nucleosome tags across thousands of genes. The position of DNase hypersensitive, nucleosome-free, sites varies between genes & is not at a fixed location with respect to the transcription start site.

Nested PCR

So you would do nested PCR as a way to deal with non-specific priming You do 2 rounds of PCR. After the first set of cycles you do a second set using the products of the first, but with new primers designed to anneal within the correct product. It is very unlikely that the second set of primers would anneal to the incorrect products from the first round of PCR. So once we have successfully made a PCR product, we want to clone it into a suitable vector. You should by now be familiar with the basic principles of DNA cloning experiments, such as inserting DNA into a simple plasmid vector. The choice of vector is going to depend on what we want to do with it. But there are certain characteristics that are common to many cloning plasmids. Brandon: I guess the disadvantage is that any errors incorporated within the body of the PCR products will be exacerbated after the second round. Basically more errors will accumulate I think in nested PCR.

Chromatin Packaging Regulating chromatin compaction

Some Histone Acetyl Transferases (HATs) themselves contain bromodomains, creating positive feedback loops - spreading of activating acetyation marks. Not all bromodomains recognize all AcLys residues - the flanking amino acids are important for recognition too. Conversely, some transcriptional repressors recruit Histone Deacetylases (HDACs) A histone code? Box 17-4 MBOG ~26 residues in the N-terminal tails of histones can be modified in various ways (acetylation, methylation, phosphorylation) - different combinations of modification signal different "activation states" of chromatin, which also depend upon the location in the genome (promoters, gene bodies, exons/ introns etc) The resultant "histone code" is more complex than just "Acetylation activates, Methylation represses" e.g. Histone H3 lysine 9 and 27 (H3K9, H3K27): di- and tri-methylation represses transcription, while mono-methylation and acetylation are activation marks. But H3K4 and H3K36 tri-methylation are associated with active chromatin.

Chromatin Packaging Regulating chromatin compaction pt 2

Some Histone Acetyl Transferases (HATs) themselves contain bromodomains, creating positive feedback loops - spreading of activating acetyation marks. Not all bromodomains recognize all AcLys residues - the flanking amino acids are important for recognition too. Conversely, some transcriptional repressors recruit Histone Deacetylases (HDACs) A histone code? Box 17-4 MBOG ~26 residues in the N-terminal tails of histones can be modified in various ways (acetylation, methylation, phosphorylation) - different combinations of modification signal different "activation states" of chromatin, which also depend upon the location in the genome (promoters, gene bodies, exons/ introns etc) The resultant "histone code" is more complex than just "Acetylation activates, Methylation represses" e.g. Histone H3 lysine 9 and 27 (H3K9, H3K27): di- and tri-methylation represses transcription, while mono-methylation and acetylation are activation marks. But H3K4 and H3K36 tri-methylation are associated with active chromatin.

Why is dsDNA sequence specific recognition by proteins a challenge?

Specific recognition is a challenge - the structure is relatively uniform, compared (for example) with the huge diversity of protein structures, and the majority of "chemical information" has been used in base-pairing...

Splicing In vitro analysis of splicing reaction

Splicing can be performed in vitro using nuclear extracts. This system was employed to isolate and analyze splicing intermediates, which in turn allowed the understanding of the splicing mechanism. 1) Synthesize splicing substrate by in vitro transcription (the substrate contains two exons and a single intron): - see Dr Scadden's lectures 2) Incubate radiolabelled RNA with nuclear extracts and ATP 3) Take samples at various times, remove proteins and analyze RNA by electrophoresis in denaturing gels (i.e. with 8M urea) followed by autoradiography 4) Note that circular intermediates migrate anomalously. Treatment with 'debranching' enzyme hydrolyses 2'-5' bonds and converts lariats to linear RNA (see below).

Splicing Tell me about the chemistry of splicing

Splicing involves two successive transesterification reactions. 1) First, the 2' hydroxyl group of the branch site attacks the phosphate on the 5' end of the intron. This leads to the release of the 5' exon and the formation of an intermediate called a lariat, in which the 5' end of the intron and the adenosine in the branch site are linked through a 2'-5' bond 2) Second, the 3' hydroxyl group of the 5' exon attacks the phosphate at the 3' end of the intron. This causes the ligation of both exons and the release of the intron in the form of a lariat 3) The lariat is 'debranched' and rapidly degraded Note that the number of phosphodiester bonds is conserved, and that no ATP is consumed during the reaction (although ATP is required for splicing, see below)

What is touch-down PCR?

Start with annealing temperatures higher than those predicted for the primers and reduce in subsequent cycles. The first successful reactions will therefore be those happening under the most stringent annealing conditions.

Tell me about Streptavidin

Streptavidin is a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14 mol/L,[1] the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton), proteolytic enzymes, and extremes of temperature and pH.

Prokaryotic Transcription Mechanism High resolution structures of RNAP complexes

Summary of points from previous slide: - β & β' pincers/jaws surround downstream DNA channel (20 bp). - Active site located at base of pincers. - FLAP (beta) and upper jaw (beta') are mobile. Holoenzyme: - σ:core interaction - 8500 Ų buried surface (x2 expected for reversible interaction). - σ regions 2, 3, & 4 interact with flexible parts of β & β'. - σ regions 2 & 4 already positioned for DNA interaction - σ 1.1 (not shown) occupies ds DNA channel - reduces non-specific DNA binding - σ3.2 linker region occupies RNA exit channel Open Complex: - σ 4 recognises -35 box in major groove by helix-turn-helix motif (cf slide 68) - σ 2 recognises -10 box on non-template strand in melted transcription bubble - shows how promoter recognised, setting correct polarity for transcription - Separate "tunnels" for template and non-template DNA in bubble. Elongation complex: - DNA strands separate at active site and turn right angle (out of page) - consistent with reduced footprint of elongation complex. - RNA exit tunnel beneath FLAP, where σ3.2 previously located

Which one is the antisense strand?

Template strand

Processing of pre-mRNA 3' end processing Tell me about Termination of Pol II Transcription

Termination of Pol II transcription Pol II transcription does not usually terminate at precise positions. Instead, it carries on for hundreds to a few thousand base pairs downstream of the mature 3' end. Evidence: Run-on transcription. 1) Incubate nuclei with NTPs and α[32P]UTP in vitro. Transcription initiation is generally inhibited, but those RNAs that are already being transcribed are completed using the labelled UTP. The newly synthesized labeled RNA molecules are cleaved to fragments of ~50-100 nucleotides, and hybridized to DNA probes spaced along the gene and downstream sequences 2) Result: There is radioactive signal well downstream of the site corresponding to the mature 3' end of the mRNA. Moreover, the signal decreases gradually in the 5'->3' direction 3) Conclusion: There are no discrete termination sites. Instead, the mature 3' end is generated via cleavage of a longer precursor and subsequent poly(A) addition

Translation in eukaryotes How do we study translation? The reticulocyte in vitro system

The 'standard' eukaryotic in vitro translation system was developed by Richard Jackson's lab in this Department and uses a lysate prepared from the reticulocytes (immature red blood cells) of anemic rabbits. The extract is prepared as follows: 1) Lyse cells (resuspend in distilled water) 2) Remove mitochondria and membranes by centrifugation (these cells are anucleate) This produces a cytoplasmic extract that will translate endogenous mRNAs (mostly α and β-globin). To remove endogenous mRNAs we need to do the following: 1) Treat the extract with micrococcal nuclease (MN) in the presence of 1mM CaCl2 (MN is completely dependent on Ca2+) 2) Add EGTA to chelate Ca2+ and inactivate the MN (EGTA is sufficiently selective for Ca2+ that the Mg2+ required for translation is unaffected) To carry out the translation reaction: 1) m7GpppG capped mRNA (in vitro synthesized by T7 RNA polymerase) 2) Reticulocyte extract 3) K+, Mg2+ to compensate for any dilution in the lysis procedure 4) ATP, GTP and a system to regenerate them (creatine phosphate and creatine kinase) 5) Amino acids including ³⁵S-Met or ³⁵S-Cys 6) Analyze by SDS-PAGE and autoradiography

What is 10x PCR buffer?

The 10x stands for the buffer concentration. 10x PCR buffer consists of 100mM Tris-HCl, pH 9.0, 15mM MgCl₂ and 500 mM KCl. Brandon: I think you have to dilute the 10x. Its just a stock buffer.

Ribosome structure and function The ribosome is a ribozyme

The 23S rRNA has peptidyl-transferase activity. Evidence: 1) Extraction of most proteins (95%) from the large subunit (50S): rRNA still has activity 2) If rRNA is damaged the activity is lost 3) 23S rRNA made by in vitro transcription can catalyze peptide bond formation (although with low efficiency) 4) Structural data: there are no proteins within 18 Å of the active site Conclusion: the ribosome is a ribozyme (i.e., a catalytic RNA molecule)

Translation in eukaryotes Initiation in eukaryotes: the scanning model

The 43S initiation complex binds first to the 5' methylated cap, and then scans in the 5'->3' direction until the first (usually) AUG is selected as the initiation site. The 60S subunit then joins and elongation starts

What is the leader sequence?

The 5' UTR is also known as the leader sequence. It is the region of an mRNA that is directly upstream from the initiation codon.

Splicing tell me about Cis sequence elements for pre-mRNA splicing (in mammals)

The 5' splice site (also called donor) is located at the exon-intron boundary 5' of the intron, while the 3' splice site (or acceptor) is present at the intron-exon boundary 3' of the intron. There are several important cis elements: 1) The majority of introns start with GU and end with AG (GU/AG rule) 2) At the 5' splice site (or donor site) the consensus sequence is (C/A)AG|*GU*RAGU. 3) The 3' end of the intron, contains three sequence elements: a) Branch point consensus, containing a conserved adenine; close to the 3' splice site (18-40 nucleotides upstream) - this sequence is very conserved in yeast introns b) Polypyrimidine tract 18-40 nt, pyrimidine rich; downstream of the branch point c) 3' splice site: Y*AG*| Consensus sequences can be displayed as pictograms, in which the size of the letter represents the frequency of the base at each position R is Pu*R*ine and Y is P*Y*rimidine

Tell me about using CRISPR to knock stuff out so you can analyze the effect of reducing the expression level of a protein

The CRISPR/cas9 system is used for genomic editing - permanent and complete changes in the genome result whereby expression of your favourite gene is altered. CRISPR/cas9 can be used to downregulate gene expression, as well as to make mutations in your sequence of interest. Genome editing uses engineered nucleases in conjunction with endogenous repair mechanisms to alter the DNA in a cell. It relies on a guide RNA (gRNA) and the bacterial Cas9 nuclease - the CRISPR/Cas system takes advantage of a short gRNA to target the bacterial Cas9 endonuclease to specific genomic loci. The CRISPR/Cas system used in gene editing consists of three components: • The Cas nuclease Cas9 (a double-stranded DNA endonuclease) • A target complementary crRNA • An auxiliary transactivating crRNA (tracrRNA). The crRNA and tracrRNA sequences are typically expressed together as a gRNA that mimics the natural crRNA/tracrRNA chimera in bacterial systems. The specific crRNA is designed using dedicated software (E.g. http://crispr.mit.edu/) - these search engines provide information about the best crRNAs to target your desired sequence. They also provide information about the likelihood of 'off-target' effects - for example, how many off-target sequences exist within coding/non-coding regions. DNA oligonucleotides encoding the crRNA are cloned into a specific site on a CRISPR plasmid to produce the gRNA as shown below: A large number of CRISPR plasmids exist to produce the gRNA - and they often also encode the Cas9 nuclease. They may also contain additional sequences for selection of cells that have been transfected with the plasmid - which may therefore be positive for genome editing (E.g. GFP, OFP, puromycin). Example: This plasmid (Invitrogen) is a pre-linearized plasmid with 5 base pair overhangs for easy cloning of your double-stranded DNA oligo that encodes a target-specific crRNA - other plasmids may have a unique restriction site for cloning crRNA. It contains the sequences necessary for producing the gRNA (i.e. crRNA+tracrRNA), the sequence encoding Cas9 (with nuclear localization signals (NLS)), and a selectable marker (orange fluorescent protein; OFP). The gRNA targets cas9 endonuclease to specific genomic loci. Cleavage by Cas9 results in doublestrand breaks in the DNA....these may get repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR)

Prokaryotic Transcription Regulation The Lac operon

The Lac operon has two promoters: - a weak constitutively active promoter that transcribes LacI mRNA, encoding the lac repressor - the regulated lac promoter - only fully switched on when Lac is available and glucose is unavailable . Glucose is an easier substrate to use, so no point in switching on Lac operon if glucose is available. This is an example of signal integration using the logic of an AND gate: IF (lactose available) AND (glucose low) OUTPUT (transcribe Lac Z,Y,A) Low basal levels of transcription mean that there is enough LacY product - permease - to allow Lac uptake into the cell when it is available. Also a sidereaction of β-Galactosidase creates allolactose (Gal-β(1-6)-Glc), which is the physiological inducer that binds to the Lac repressor (see next few slides) The regulated promoter has suboptimal -10 and -35 elements (indicated by lower case), meaning that maximal levels of transcription requires an activator, the CAP protein. The "Operator" - binding site for Lac repressor (palindromic) - overlaps the transcription start site/RNAP footprint Binding site for CAP protein (palindromic) is upstream of -35 box, but not overlapping

Prokaryotic Transcription Mechanism Bacterial core promoter elements are asymmetric

The LacUV5 promoter is shown in the "open complex" form with a transcription bubble from -10 to +2 (more on this in a couple of slides). Bacterial promoters have two main consensus sequences at -10 and -35. Mutations (natural or experimentally induced) at these sites can alter transcription efficiency Note that both the -10 and -35 consensus sequences are asymmetric - no direct or inverted repeats. These elements are recognised by RNAP sigma subunit, setting the polarity for transcription A third consensus element (UP) is present only at very strong promoters Apart from these elements, sequences are not conserved between different promoters except at +1 (A) The spacing between -10 and -35 elements is conserved; in >95% of E coli promoters it's between 16-18 bp - the relative orientation of the -10 and -35 elements needs to be maintained for recognition by RNAP Note that although sequences outside -10 and -35 boxes are not conserved, footprinting and modification-interference extends beyond these specific sequences. Moreover, the patterns of footprinting & modification interference are similar on different promoters even though the sequence varies. Although not shown on the slide, T's in the transcription bubble at -10 to +2 are sensitive to KMnO4 oxidation. phosphate ethylation - ethyl group binds to phosphates interfering with protein binding to a specific site.

Regulation of translational efficiency in bacteria Effect of RNA secondary structure on the accessibility of the SD sequence

The MS2 coat protein gene (B protein) has the SD sequence folded into a secondary structure. The following experiment addressed the role of this structure on the control of translation: 21 different point mutations were made in the coat protein gene that destabilized or stabilized the structure around the SD sequence, but did not change SD or the encoded protein sequence. The translation efficiency was measured in vivo. From established RNA folding rules it is possible to calculate ∆G, and hence Keq for the folded vs unfolded conformation, and hence the fractions of unfolded molecules. A plot of the fraction of unfolded mRNA vs. translation efficiency shows good correlation, demonstrating that the accessibility of the SD sequence modulates translation initiation

Regulation of translational efficiency in bacteria Translational control by translational repressors: the MS2 coat protein

The MS2 coat protein itself binds with high affinity to the initiation site of the replicase gene. This association stabilizes a stem-loop structure that represses translation initiation. Therefore, once the coat protein accumulates, it blocks the synthesis of more replicase. This ensures that the replicase gene is translated only during the early phase of infection

Internal initiation Introduction and importance Tell me about Pico RNA viruses

The Picornaviruses are RNA viruses responsible for polio, common cold and other conditions. Their mRNA is uncapped and has a long 5' UTR (~600-1200 nucleotides) with many AUG codons and extensive stable secondary structure. Both features should prevent access to the authentic AUG by a scanning ribosome. This mRNA is not translated by ribosome scanning, but by direct ribosome binding to an internal ribosome entry site or IRES. Several picornaviruses produces a protease that cleaves and inactivates eIF4G, leading to a shutdown of cellular protein synthesis. The use of IRES allows viral proteins to be translated in a cap-independent way picornavirus IRESs do not bind the 40S subunit directly(some IRESs do), but are recruited instead through the eIF4G-binding site

How are nucleotide bases linked to the sugar of nucleic acids?

The bases - linked to the sugar C1 via N-glycosidic bonds to N1 of pyrimidines or N9 of purines

How do you estimate the density of bacteria in culture?

The density of bacteria in a culture can be estimated by measuring the apparent absorbance (technically turbidity due to the light scattering) at 600 nm in a spectrophotometer.

Regulation of translational efficiency in bacteria Introduction

The efficiency of initiation of translation in bacteria is partly controlled by the pairing potential of the SD sequence to the 16S rRNA. However, the correlation is not perfect: a) Initiation efficiency of some mRNAs is unrelated to the fit of their SD sequence to consensus b) Mutations close to but outside the SD sequence or AUG, can have a major effect These results can be explained by the fact that the accessibility of the SD sequence can be regulated by the direct binding of regulators (proteins or sRNAs) or by the presence of RNA secondary structures. In turn, mRNA secondary structures - and thus translation - can be modulated in several ways: a) Translation of other ORFs in polycistronic mRNA (translational coupling) b) Proteins c) Temperature (thermosensors) d) Non-coding RNAs (sRNAs) e) Small molecules (riboswitches) RNA bacteriophages illustrate several interesting aspects of translational regulation. The MS2 phage mRNA contains four genes: A (maturation protein), B (coat protein), C (RNA replicase) and D (lysis gene, which overlaps B and C in the +1 reading frame) We will use this mRNA to illustrate how secondary structure regulates translation, and how translation and repressor proteins control translation through their effect on RNA secondary structure 1) Effect of RNA secondary structure on the accessibility of the SD sequence 2) Translational coupling 3) Translational control by translational repressors: the MS2 coat protein 4) Regulation of gene expression in bacteria by small non-coding RNAs (sRNAs) 5) Regulation of translation by thermosensors 6) Termination of transcription in bacteria 7) Attenuation

Processing of pre-mRNA 5' end processing: capping Tell me about Cap formation and function

The formation of the cap takes places cotranscriptionally in several steps: 1) The γ phosphate from the 5' tri-phosphate end of the pre-mRNA is removed by RNA terminal phosphatase (RTPase) 2) GMP is transferred from GTP by an RNA guanylyl transferase (RGTase), producing G5'ppp5'N and releasing pyrophosphate 3) The added guanine is methylated at position 7 by RNA-(guanine-N-7)-methyltransferase. The methyl donor is S-adenosylmethionine. This structure is called Cap 0. In yeast this is the only modification, while in mammalian cells some nucleotides on the mRNA are also modified 4) Capping enzymes (RTPase and RGTase) are part of the same polypeptide in multicellular organisms (in yeast they are separate, but they form a heterodimer) 5) Capping is a very early cotranscriptional event 6) The cap is bound in the nucleus by a dimer called cap-binding complex (CBC); in the cytoplasm it is bound by other proteins (see translation)

Tell me about the lacZ' gene of pBluescript

The lacZ' gene encodes β-galactosidase, which enables 'blue-white' selection. While this is not essential for cloning, it is extremely useful for efficiently selecting the correct clones. The lacZ gene is rather large the Mr of LacZ is 116,000 - how big would the gene be?), so only a fragment of the lacZ' gene (encoding the first 131 amino acids of β-galactosidase) is actually encoded within the pBluescript II vector. The remainder of the lacZ gene is encoded within the host DNA (on the F' plasmid; see later) - together the part of lacZ encoded on pBluescript and the bit within the host genome are able to assemble and give rise to functional β-galactosidase: Production of β-galactosidase in E. coli enables cells containing it to break down an artificial substrate, X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) to produce a blue pigment. This is the basis of blue-white selection: The lacZ sequence in pBluescript (and other cloning vectors) is interrupted by the MCS (see above). So if a DNA sequence is cloned into the MCS, the lacZ' gene will be disrupted, which renders β-galactosidase dysfunctional and therefore results in white colonies. In contrast, in the absence of an insert, the lacZ' gene will be functional and the colonies will be blue. I.e.

Regulation of translation in eukaryotic cells

The main point of control is initiation. Translational regulation can be global (affecting most mRNAs in the cell) or transcript-specific. The former is often controlled by modulating the activity of general translation factors, while the latter is regulated by sequence-specific RNA-binding proteins. The two steps that are targeted most commonly in global control are the recycling of eIF2B and the interaction between eIF4E and the cap.

Tell me about pBluescript?

The plasmid vector into which we cloned our POU domain fragment . It has important features (on the picture) MCS is a cluster of restriction enzyme recognition sites, which in this plasmid includes sites for BamHI and HindIII. Cut the PCR product and the vector with both these enzymes so they will have complementary sticky ends, which should anneal to each other. Then you must ligate it. Successful ligation means the resultant plasmid will have lacZ' gene interrupted by the extra piece of DNA. The success of the ligation can be monitored by an assay for lacZ'. You will use a modified version of the sugar galactose called 5-bromo-4-chloro-3-indoyl beta-D-galactopyranoside (abbreviated to X-gal), which when cleaved by B-galactosidase gives rise to a blue product.

Tissue-specific knockouts can also be generated using the Cre system. How would you do that?

The problem is that if you make heterozygotes and cross them you never recover any homozygotes, as loss of the protein throughout the organism is lethal. But if we can cause loss of the protein in specific tissues, we may be able to get round that problem. In this case (Uhlmann EJ et al., (2002) Annals of Neurology 52: 285-296), they wanted to knock out the Tsc1 gene in astrocytes. The stages are as follows: 1) Replace the endogenous Tsc1 gene with one that is flanked by lox sequences (or has one or more exons flanked by lox sequences). This can be done using ES cells as described. First you make mosaics, then get heterozygous non-mosaics and then cross those to get homozygotes. Note that in the absence of Cre recombinase, the lox sequences are inactive, and these strains behave like wild type. The homozygotes are designated Tsc1c/c, indicating they are homozygous for a conditional Tsc1 mutation. 2) Separately make homozygous transgenic mice with a sequence inserted containing the Cre recombinase gene under the control of a promoter active in astrocytes. (The authors of this paper used GFAP - glial fibrillary acidic protein.) 3) Cross the two mouse strains. The progeny will be heterozygous for the Tsc1 conditional mutant allele and for the introduced GFAP-Cre allele. (This will result in the expression of Cre in their astrocytes and thus the inactivation of the floxed copy of Tsc1 in astrocytes. Don't bother about that. Their germ line is fine.) They are Tsc1^(c/+); GFAP-Cre/+ 4) Cross the Tsc1^(c/+); GFAP-Cre/+ individuals to Tsc1^(c/c); +/+ homozygotes. The progeny will be as follows (we can distinguish them by extracting DNA from tissue samples and checking which alleles they have by PCR): Tsc1^(c/+); GFAP-Cre/+ these are not what we want Tsc1^(c/+); +/+ these are also not what we want Tsc1^(c/c); +/+ these are also not what we want Tsc1^(c/c); GFAP-Cre/+ These are what we want. They are homozygous for the floxed Tsc1 allele. The Cre recombinase is expressed from the GFAP promoter in astrocytes, leading to excision across the lox elements in both copies of the Tsc1 gene. So they are homozygous null for Tsc1 in astrocytes, but not elsewhere.

Translation elongation in prokaryotes Introduction

The ribosome contains three tRNA binding sites: A, P and E. The P site contains the peptidyl-tRNA (or Met-tRNAf if translating the first codon), with the tRNA paired to last translated codon. The A site contains the next aminoacyl-tRNA to be added to the growing peptide, with the tRNA paired to the next codon to be translated. Any aminoacyl-tRNA can enter the A site except the initiator. The process of elongation involves three steps: 1) Binding of the aminoacyl-tRNA to the A site 2) Formation of the peptide bond by the transfer of the polypeptide from site P to the aminoacyl-tRNA in site A 3) Translocation of the ribosome a) The new peptidyl-tRNA is transferred to the P site b) The deacylated tRNA leaves the P site and is ejected through the E site The process of elongation requires the action of three elongation factors: EF-Tu, EF-G and EF-Ts. EF-Tu brings the aa-tRNA to the A site, EF-G is required for translocation, and EF-Ts is necessary for the recycling of EFTu

Translation The ribosome cycle

The ribosome cycle 1) The small subunit associates with the mRNA first, followed by the large subunit. After that, both subunits are stably associated with each other. They can be dissociated (irreversibly) by unphysiological treatments such as EDTA 2) At termination the two subunits are released separately 3) When not translating mRNA the two ribosomal subunits can reversibly associate depending on Mg2+ and K+ concentrations. Under physiological conditions, equilibrium is strongly in favour of association 4) The binding of a 'dissociation factor' to the small subunit prevents reassociation of the two subunits. The dissociation factor — initiation factor IF3 (prokaryotes) or eIF-3 (eukaryotes) — also primes the small subunit for initiation 5) IF-3/eIF-3 is released during initiation 6) IF-3/eIF-3 is present at about 1 molecule per 10 ribosomes. Therefore, only about 10% of ribosomes can be in dissociated form. The rest must be in translating polysomes or present as inactive 70S or 80S ribosomes Remember the ribosome has three tRNA binding sites, called A, P and E

When analyzing gene expression tell me about using qPCR

The simplest thing is to do PCR and run the products on an agarose gel (typically stained with Ethidium Bromide). More template - more products. But difficult to quantify due to saturation if using many PCR cycles. Test yourself E: "More template - more products" that makes certain assumptions - what might they be? But there are more quantitative methods that allow you to detect product accumulation in real time.... a. Using SYBR green: One (very widely used) is to include a dye that binds to double-stranded DNA and fluoresces - E.g. SYBR green. The intensity of fluorescence generated by SYBR green above background is measured, and the number of PCR cycles required to generate fluorescence above a particular threshold is referred to as the CT value. This reflects the relative amount of newly generated double-stranded DNA strands b. Using a sequence-specific fluorescent probe: A more specific method is to include a specialised oligonucleotide probe that hybridises to a region within the target sequence. • The probe is labelled at the 5' end with a fluorescent reporter, and at the 3' end with a quencher. • When the reporter and the quencher are close, i.e. on the same molecule, get no fluorescence. • The probe anneals to target DNA, and the 5'-3' exonuclease activity of the DNA polymerase cleaves the 5' label from the probe, thus allowing fluorescence.

Chromatin Packaging Genome-wide Nucleosome mapping

The structure of the nucleosome, with the numerous sequence non-specific protein-DNA contacts is consistent with the general packaging role. However, positioning of nucleosomes can be regulated. The figure on the previous slide shows the density of Pol II (from chromatin immunoprecipitation followed by sequencing - slide 47) and of nucleosomes relative to transcription start sites (TSS). Nucleosomes were mapped by treating chromatin with micrococcal nuclease (slide 75) to generate 147 bp fragments. Deep-sequencing of these fragments maps the positions of nucleosomes, genome-wide. The experiment was carried out in resting and activated T-cells. The Figure shows the average relative density of nucleosomes and of RNA Pol II around TSS's, averaged across thousands of genes. As expected, RNA Pol II is present at much higher levels on expressed genes (green dashed line) than non expressed genes (pink dashes). The strong peak of RNA Pol II at the TSS of expressed genes is commonly observed and reflects the fact that promoter escape can be rate limiting. Nucleosomes are present on both expressed (red line) and non-expressed (blue line) genes. Non-expressed genes do not show a generally higher level of nucleosomes. However, the blue line is "flatter" than the red line, indicating that nucleosomes are not strongly positioned on non-expressed genes. The expressed genes show a distinct peak at + 40 and a lower density of nucleosomes upstream of the TSS (i.e. at the promoter). This preferential positioning of nucleosomes arises from their exclusion from regions where the transcription machinery binds at the promoter.

Methods to study posttranscriptional processes Tell me about Single-gene view and genome-wide analysis

The use of individual 'model' transcripts allows the detailed investigation of the molecular mechanisms underlying posttranscriptional processes. These approaches can be complemented with methods that provide a genome-wide view of these processes. These methods can deliver information on how general the molecular findings obtained with single genes are, and also give insight into how gene expression is coordinated for groups of genes. Genome-wide approaches require the ability to identify and quantify thousands of molecules of nucleic acid in parallel. The main tools are DNA microarrays and deep sequencing (also known as massive parallel, high-throughput, or next-generation). See IA lectures by Dr S. Russell for more details. Below are some examples of what can be achieved with these methods: 1) Systematically identify targets of RNA-binding proteins 2) Measure the use of different exons and introns 3) Determine decay rates for all mRNAs in a cell 4) Measure translation rates for all mRNAs

What is a western blot?

The western blot (sometimes called the protein immunoblot) - detects specific proteins in a sample of tissue extract. In brief, the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognises and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognises and binds to the primary antibody. The secondary antibody is visualised through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein.

Termination of translation in prokaryotes

There are no tRNAs for termination codons. Instead, proteins called release factors (RFs) recognise termination codons and induce the hydrolysis of the growing peptide chain from the tRNA. There are two types of termination factors: 1) Class I: a) Recognise the termination codon directly b) There are two: RF1 recognises UAA and UAG and RF2 binds to UGA and UAA c) Induce hydrolysis of the peptidyl-tRNA 2) Class II: a) Single factor (RF3), which is a GTP-binding protein with GTPase activity b) RF3 binds the ribosome in the GDP-bound form c) GTP replaces GDP while on the ribosome, leading to a conformational change that releases the class I factor d) Subsequent GTP hydrolysis allows the release of RF3 See below for a detailed discussion of the functions of the RF1 and RF2 proteins

What other considerations do you need to think about when making primers apart from i) polymerase errors ii) size of product and iii) non-specific priming?

There are other considerations you also need to think about for making primers - • Ideally 20-30 bp in length • Avoid mismatches at 3' end • Optimal to have a C or G at the 3' end to get efficient extension (3 H-bonds) • Annealing temp should be similar for both primers • Internal secondary structure should be avoided • Complementarity between primers should be avoided - can get primer dimers forming (and primers are present in excess, so even if primers aren't completely complementary to each other, they are simply more likely to bind to each other because excess) With PCR you need to be wary of contamination

Identifying interacting proteins

There are various methods that can be used to look for protein-protein interactions - your method of choice may depend on the available reagents, ease of expression of your protein, or what questions you are trying to address etc. In addition, there are various methods that can be used to identify interactions between proteins and a range of ligands etc. 1) Immunoprecipitations 2) 'Pull-down' assays using fusion proteins 3) Yeast two-hybrid system

You want to make recombinant proteins. What stuff do you need to add to your plasmid so this can happen? Tell me about some commonly used conventional E coli promoters

There are various systems available to produce recombinant proteins. The most commonly used of these are E. coli, which we will now consider. Expression in E. coli will require: • Promoter (preferably strong and controllable) • Prokaryotic ribosome binding site • AUG initiation codon. • and, possibly, transcription termination. Commonly used "conventional" E. coli promoters include: • PL from phage lambda; controlled by cI857 • PlacZ; controlled by LacI protein, and IPTG • Ptac; a hybrid between the -35 region of the trp operon and the -10 region of the lac operon, which is very powerful. It can still be controlled by the lac repressor and IPTG. • Ptrc - similar to Ptac. • T7 - Transcribed by T7 RNA pol - which is itself regulated by lac promoter (IPTG) - see below: The promoter from bacteriophage T7 is widely used, but this requires expression of the corresponding polymerase in the cell. This is usually achieved in a two-stage expression system. The gene to be expressed is placed under the control of the T7 promoter. The host contains a gene for the T7 polymerase under the control of the lac promoter. So you add IPTG, which induces synthesis of the T7 polymerase and leads to transcription of the gene of interest A widely used example is the pET vector system. The host for expression is typically based on E. coli strain BL21, which has been made lysogenic for a modified lambda phage which carries the T7 polymerase under the control of a lac promoter.

Tell me about the Multiple Cloning Site of pBluescript

There is a collection of recognition sites for restriction enzymes (called the multiple cloning site, or MCS) that are located within the lacZ' gene in pBluescript. In pBluescript II (+/-) KS, the MCS is in the other orientation (i.e. The KpnI restriction site is nearest the lacZ promoter and the SacI site is farthest from the lacZ promoter) - may be useful for some applications depending on cloning strategy.

Translation in eukaryotes Initiation in eukaryotes: mechanism and factors

There is no need to remember all factors in the figure below, only those discussed in the lecture! Initiation involves three major steps: 1) Formation of the 43S initiation complex: this requires the 40S ribosomal subunit, initiation factors and initiator tRNA. This step is independent of mRNA 2) Formation of the cap-binding complex at the 5' end of the mRNA 3) Assembly of the 48S initiation complex: the 43S complex joins the cap-binding complex on the mRNA 4) The formation of the 43S initiation complex requires several initiation factors, including: a) eIF3, which prevents reassociation of ribosomal subunits (as IF3, see above) b) eIF2, which is a trimeric GTP-binding proteins (bacterial IF2 is monomeric) that brings the initiator to the small subunit 5) The formation of the cap-binding complex involves the following factors: a) The eIF4F complex, which recognizes the cap first. It is made up of eIF4A, eIF4G, eIF4E. eIF4G is a scaffold protein (it binds the 40S subunit, eIF4A, eIF4E and eIF3), eIF4A is a helicase, and eIF4E recognises the cap b) eIF4B joins the cap after eIF4F; it stimulates eIF4A activity

We assume the probability of a cell taking up two plasmids is very low indeed, but that may not be valid. Why not?

There might be a sub-population of cells that's very good at picking up DNA

Pull down assays using fusion proteins

This assay used for purification and other applications downstream.... It is an in vitro method used to determine a physical interaction between two or more proteins. This method is only possible if your protein of interest can be expressed as a soluble protein. You can make use of tags such as GST or His-tags to make a fusion protein that can be immobilised on a column. E.g. A GST-tagged protein can be immobilised using Glutathione sepharose. A complex mixture of proteins can then be passed over the immobilised protein, and hopefully stick if protein-protein interactions occur. I.e. • Immobilise your GST-tagged protein on glutathione sepharose • Add the complex mixture of proteins to the column • Wash unbound proteins from column • Elute GST-tagged protein of interest (and interacting proteins) from column using glutathione • Analyze on SDS-PAGE, then use mass spectrometry to determine the identity of interacting proteins • Verify interactions using Immunoblots E.g. Purifying proteins that interact with GST-ADAR1 (GST-A1):

What is 6x Loading Buffer?

This contains glycerol to make the sample dense and stop it diffusing out of the well into the buffer, and a blue dye (bromophenol blue) to help you judge how far the electrophoresis is going. You use 6x Loading Buffer when checking the size of the PCR amplified DNA on a 0.8% agarose gel. • LB - 6x Loading Buffer: 60 mM Lithium acetate, 60 mM Boric acid [I think this is a buffer) (final concentration 10 mM Lithium acetate, 10 mM Boric acid, pH ~6.7), 60% (v/v) Glycerol, 0.03% (w/v) bromophenol blue

Cloning PCR products into a vector: 5.4 Gibson's assembly method

This is a method by which you can join almost any 2 fragments regardless of sequence. In fact, multiple (>10) overlapping DNA fragments can be joined at once in a single isothermal reaction to get a fully ligated double stranded DNA molecule. This method has the advantage that there is no need for specific restriction sites, that there is no 'scar' between joined fragments, and that cloning is both directional and efficient (multiple sequences joined in a 1 h reaction). Example: you could assemble 5 PCR fragments (E.g. 4 inserts, 1 plasmid) together in a single reaction. So the basis of this method is that the fragments must have overlapping ends that are used for directional assembly of fragments.

Tell me about lacUV5 promoter

This is a mutant form of the lacZ promoter. It carries point mutations in the -10 region of the promoter, which increase its efficiency.

Why would sequence specific recognition of a complementary dsRNA molecule be challenging?

This is because W-C base-paired dsRNA has an A form helix, this means the information rich major groove would be inaccessible.

Regulation of translation in eukaryotic cells Transcript-specific regulation of translation

This regulation is performed by sequence-specific RNA-binding proteins (RBPs) that recognise cisacting sequences and modulate translation. In most cases the binding sites are in the 3' UTRs, but there are exceptions 1) Repression at the 3' end: general mechanism a) Three proteins involved (X, Y, Z) b) X: sequence-specific RBP that binds to the 3' UTR and confers transcript specificity c) Z: cap-binding protein (usually a eIF4E isoform) d) Y: bridging protein, interacts with both X and Z e) The interactions among X, Y and Z lead to the formation of a loop that blocks translation by inhibiting eI4F recruitment

Processing of pre-mRNA 5' end processing: capping Give me an introduction to Pol II CTD

To understand the specificity of capping we first need to look at the function of the carboxyterminal domain of the large subunit of RNA polymerase II (Pol II CTD) 1) CTD contains a linker followed by tandem repeats of a 7 amino acid sequence (consensus is YSPTSPS) 2) The repeats contain multiple residues that can be phosphorylated 3) No analogous structure is present in Pol I or Pol III 4) Sequence conserved from yeast to mammals 5) 26 repeats (yeast) / 52 repeats (mammals) 6) Domain is unstructured 7) Located close to the exit channel (good position to recruit enzymes that modify mRNA) 8) CTD is phosphorylated/dephosphorylated during transcription (by different kinases) a) Initially dephosphorylated b) Early phosphorylation in S5, later removed c) Later phosphorylated in S2 9) Proteomic analysis (in yeast) identified >100 proteins associated with phosphorylated CTD; the CTD functions as a 'landing platform' for factors involved in cotranscriptional processes

What is transfection?

Transfection is the process of deliberately introducing naked or purified nucleic acids into EUKARYOTIC cells. There are two possible results : Stable and transient transfection

Translation Comparison between prokaryotes and eukaryotes

Translation is generally quite similar in both systems 1) Eukaryotic ribosomes are larger and more complex. 2) Translation initiation is very different in both systems, and requires a much more complex machinery in eukaryotes (see table)

Translation Intro

Translation is the process of protein synthesis from an mRNA template. Translation requires activated tRNAs, ribosomes and a number of ancillary factors (translation factors). The table below lists the main components involved in translation:

GTP hydrolysis and translation

Translation makes widespread use of GTPases (IF2, EF-Tu, EF-G, RF3). However, GTP hydrolysis is not coupled to chemical modifications. Instead, GTP hydrolysis is used to ensure the correct order and fidelity of the process. An example of this function is provided by EF-Tu: 1) The conformation of EF-Tu changes dramatically between the GDP and GTP-bound forms 2) Only the GTP-bound form is capable of binding aa-tRNAs, masking the aminoacid and preventing it from reacting with the peptidyl-tRNA (BL: because aren't sure yet if aa-tRNA is the right one, so you wanna mask it to prevent the aa from adding onto the growing peptide chain UNTIL you are sure that its the right amino acid). 3) The GTPase activity of EF-Tu is only stimulated when the codon-anticodon pairing is correct; GTP hydrolysis and EF-Tu release allow the formation of the new peptide bond 4) If the wrong aa-tRNA enters the A site it does not activate EF-TU GTPase activity, and is released without reacting with the peptidyl-tRNA 5) In this way, the new peptide bond can only form when the correct tRNA is in the A site

Tell me about Type II Restriction Enzymes

Type II Restriction Enzymes Three types of restriction enzymes are known, and the ones you are familiar with are type II. Types I and III do not cut in the recognition sequence, and are less useful, as you can't predict what "sticky ends" they will leave on the fragments generated. There are other differences, too. Note that different enzymes can give the same sticky ends (E.g. BamHI and BglII). However, if fragments with those ends are joined, neither recognition site is recreated. PCR can be used to create DNA fragments with unique restriction sites on the ends by incorporating restriction sites into the primers (make sure those restriction sites are not contained within the DNA to be cloned!).

Primer design... You need to have some sequence information before you do PCR.... 2. You may be able to obtain the sequence directly using genomic data......

Use online resources to find your sequence. § There are a large number of genome projects for different organisms in which complete genomes or sets of cDNA sequences are sequenced. Also, in the context of genome sequencing projects, it's common to assemble a collection of cloned cDNAs and determine the 5' or 3' end of the sequence first - referred to as Expressed Sequence Tags (ESTs). ESTs are typically >400 nt in length, and may be prepared from different tissues/developmental stages. Many EST databases are commercially available. (E.g. I.M.A.G.E. clones - Source Biosciences (http://www.lifesciences.sourcebioscience.com/cloneproducts/mammalian/cdna/image/)) § Various web databases are available - E.g. NCBI (https://www.ncbi.nlm.nih.gov/) -where you can search for your sequence of interest. o E.g. using the name of your protein o Using BLAST (Basic Local Alignment Search Tool) if you have a limited amount of sequence (either nucleotide or amino acid sequence). You can use BLAST to search for more sequence from the same organism as the query sequence, or for homologous sequences from other organisms. You can search nucleotide databases using a nucleotide query (nucleotide blast), protein databases using a protein query (protein blast), protein databases using a translated nucleotide query (blastx), translated nucleotide databases using a protein query (tblastn) etc. § Other genome browsers have additional information that can be useful when designing primers - E.g. information about gene structure, isoforms etc. o E.g. UCSC Genome Bioinformatics (http://genome.ucsc.edu/index.html) o E.g. Ensembl (http://www.ensembl.org/index.html ) So online databases make it very efficient for obtaining the sequence of interest, which can then be used to design PCR primers. PCR is then used to amplify the sequence of interest using genomic DNA or cDNA sequences as a template. Alternatively, it is sometimes possible to order a clone containing the sequence of interest using information from the various web databases - E.g. I.M.A.G.E clones that comprise either ESTs, partial or potentially full coding sequences may be commercially available, which can then be ordered directly (E.g. Source BioScience).

Prokaryotic Transcription Mechanism NAD+ cap can stabilize prokaryotic mRNAs

Very recently it has been found that some transcripts have an NAD+ cap at their 5' end, rather than the usual triphosphate end. Unlike eukaryotic mRNAs where the cap is added by other enzymes - either post- or co-transcriptionally, in prokaryotes NAD+ can compete with ATP for direct incorporation by RNA polymerase at the +1 position. This is because part of the NAD structure contains adenosine diphosphate. The specific promoter sequence determines whether NAD+ can be incorporated. NAD+ capped transcripts are much more stable than other prokaryotic mRNAs. This is therefore a novel mechanism for regulating gene expression in bacteria.

What is Virkon?

Virkon is a multi-purpose disinfectant. It contains oxone (potassium peroxymonosulfate), sodium dodecylbenzenesulfonate, sulfamic acid, and inorganic buffers. It is typically used for cleaning up hazardous spills, disinfecting surfaces and soaking equipment. The solution is used in many areas, including hospitals, laboratories, nursing homes, funeral homes, dental and veterinary facilities, and anywhere else where control of pathogens is required. Virkon has a wide spectrum of activity against viruses, some fungi, and bacteria. However, it is less effective against spores and fungi than some alternative disinfectants

What is Coomassie Brilliant Blue G250

Was used when purifying POU domain. After the affinity purification, you will perform a Bradford assay to estimate the concentration of protein that you have produced. use dye Coomassie Brilliant Blue G250, which is green-brown when free in acid solution, and becomes blue upon binding to protein. use spectrophotometer to measure the intensity of the blue colour developed by a series of protein solutions of known concentration. allows standard curve to be constructed The reaction is based on the Coomassie dye, which changes colour when it binds to basic amino acids (mainly arginine) within proteins. This reaction is affected by the presence of detergent, and also by the composition of the protein in question (i.e. how many arginine/hydrophobic groups it has). The relationship between A₅₉₅ and concentration is also not linear at higher concentrations.

Ribosome structure and function

We now have high-resolution structures of ribosomes at different stages of translation and in complex with different translation factors, tRNAs and antibiotics. The Nobel Prize for Chemistry in 2009 was awarded to Ramakrishnan (Cambridge), Steitz and Yonath for studies on ribosome structure. The structures confirmed and complemented previous biochemical studies. The structure of the ribosome is organized around the rRNA, which represents over 60% of the ribosome mass. rRNAs form very complex secondary and tertiary structures (~60% of bases form standard Watson/Crick pairs). The majority of the proteins contact the rRNA directly, but there are very few protein-protein interactions. An important fact revealed by the structures is that the interface between subunits is almost completely devoid of proteins. You can watch the picture above as well as movies of the structure of the ribosome at the website of the Noller lab (http://rna.ucsc.edu/rnacenter/ribosome_images.html) rRNA tertiary structures are stabilised in several ways: 1) Interactions with proteins 2) Standard long-range base pairs between nucleotides distant on the secondary structure 3) Unconventional nucleotide-nucleotide interactions Many ribosomal proteins have a globular domain and long extensions that would be disordered in isolated proteins. These extensions are often buried in the RNA and are crucial for RNA folding The structure of the ribosome reveals the location of the A, P and E sites, which are located between both subunits. The pairing between mRNA and anticodon takes place in the small subunit, while the peptidyl-transferase site (PTS) is located in the large one

Analysing the effect of reducing the expression level of a protein... 3 different ways of knocking out gene function?

We often want to reduce expression of a protein to verify its function, or to look at the phenotype in its absence. There are various ways we can knock-down or knock-out gene function: A. RNA interference (RNAi) - post transcriptional gene silencing B. CRISPR/cas9 C. Gene inactivation by homologous recombination

Regulation of translational efficiency in bacteria Regulation by riboswitches guanine-binding riboswitch

We will use the guanine-binding riboswitch to illustrate how riboswitches can detect specific ligands. This riboswitch binds to guanine with high specificity (a 10,000-fold higher affinity for guanine than for adenine) 1) The guanine riboswitch forms a secondary structure made up of three stems (P1, P2 and P3), two loops (L2 and L3) and three junction regions 2) The riboswitch folds into a tertiary structure in which the three helices are parallel to each other. This structure is stabilized by base-pair interactions between the three stems (including base triples) 3) The ligand-binding pocket is made at the junction between the three stems. In it, two bases interact directly (by forming hydrogen bonds) with the ligand 4) The tertiary structure is essential for ligand recognition. If the L2/L3 interaction is disrupted there is a ~1,000 fold decrease in affinity for guanine The guanine riboswitch regulates gene expression by controlling attenuation 1) In the presence of guanine a terminator is formed, leading to premature transcription termination 2) In the absence of guanine, P1 does not form and the structure does not contain a terminator page 33 mata for clearer picture

"More template - more products" that makes certain assumptions - what might they be?

We're assuming that the amount of product is determined entirely by the amount of starting material. That means that everything else is in excess - primers, dNTPs etc, and that no degradation takes place. Those assumptions may not be valid - but you could include some standards with known amounts of starting template over a range that included the samples you need to measure.

Safety of acrylamide?

Wear gloves when handling gels. acrylamide is absorbed through the skin and is neurotoxic

Wanna see A form, B form and Z form DNA?

You can only say yes ;)

Measuring sizes of DNA molecules: GelRed and Ethidium Bromide?

When running a gel, ethidium bromide traditionally is added. This is a flat polycyclic molecule capable of fitting between the bases in DNA (intercalation). Intercalated dye fluoresces much more strongly under UV light than free dye, so the position of DNA molecules in your gel can be easily determined. GelRed can also be used to stain the nucleic acids in the gel. GelRed in structurally closely related to ethidium bromide and consists of two ethidium subunits that are bridged by a linear spacer. Its a fluorophore and therefore its optical properties are essentially identical to those of ethidium bromide. When exposed to UV it will fluoresce with an orange colour that strongly intensifies after binding to DNA. The substance is less toxic and more sensitive alternative to ethidium bromide. Picture is Ethidium Bromide

What is X-gal

X-gal is an analog of lactose, and therefore may be hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose. X-gal, when cleaved by β-galactosidase, yields galactose and 5-bromo-4-chloro-3-hydroxyindole - 1. The latter then spontaneously dimerizes and is oxidized into 5,5'-dibromo-4,4'-dichloro-indigo - 2, an intensely blue product which is insoluble. X-gal itself is colorless, so the presence of blue-colored product may therefore be used as a test for the presence of active β-galactosidase. This easy identification of an active enzyme allows the gene for β-galactosidase (the lacZ gene) to be used as a reporter gene in various applications

Is a bacteriophage a virus?

YES

Tell me about using reverse transcription when analyzing gene expression

You can use RT to estimate the abundance of a particular mRNA in a sample. This is usually done by having a round of reverse transcription, using a reverse transcriptase enzyme and a single primer, to make a single strand of cDNA prior to the PCR itself. The primer for reverse transcription could be oligo-dT for general cDNA synthesis from polyadenylated messages, or it could be specific to a particular message. Uses Reverse Transcriptase- E.g. isolated from avian myeloblastosis virus (AMV) or Moloney murine leukaemia virus (MMLV). Various modified reverse transcriptase enzymes are available - they are optimized for better yields (E.g. Superscript® II Reverse Transcriptase is based on MMLV, but has increased thermal stability and reduced RNase H activity - this eliminates degradation of RNA molecules during first-strand cDNA synthesis and therefore gives better yields). E.g. Superscript II - has superior performance characteristics: o Yield—reduced RNase H activity results in greater first-strand cDNA yields o Size of cDNA—generation of RT-PCR products up to 12 kb o Thermostability—full activity at 42 °C (normal temp for RT reactions) o Flexibility—uses either total or poly(A)+ RNA First strand synthesis is sufficient for RT-PCR - the products are used for PCR. Sometimes want to synthesise other strand for some applications.... and there are various ways to achieve that - E.g. RNase H method.

How would you make a gene to synthesize dsRNA?

You could make a double stranded RNA inside the cell (rather than putting the dsRNA directly into the cell) by making an artificial gene that contained the target sequence in an inverted repeat configuration. Transcription of the gene would produce an RNA that was self-complementary. (This is analogous to antisense RNA, except that a single hairpin RNA molecule is used, rather than the two separate RNAs as in antisense.)

Analyze gene expression...

You may want to know something about how mRNA levels change over time, or in different tissues, or at different developmental stages etc. One relatively straightforward and sensitive method for quantifying mRNA levels is using Reverse Transcription and Quantitative PCR (qPCR) (RT/qPCR (pp47-48)). We can amplify mRNA sequences as DNA copies (equivalent to cDNA cloning) using RT/qPCR. It's like ordinary PCR except the PCR reactions are preceded by a replication reaction at normal temperatures with a reverse transcriptase enzyme. The amount of DNA product produced using RT/qPCR correlates to the amount of mRNA that we are started with. 1) Reverse Transcription (RT) 2) Quantitative PCR The simplest thing is to do PCR and run the products on an agarose gel (typically stained with Ethidium Bromide). More template - more products. But difficult to quantify due to saturation if using many PCR cycles. a. Using SYBR green: b. Using a sequence-specific fluorescent probe:

Why would you treat a plasmid with alkaline phosphatase?

You treat the ends with alkaline phosphatase, to remove the 5' phosphate groups and reduce the chances that the plasmid will ligate to itself(intramolecular ligation). Removing terminal phosphate groups leaves only hydroxyl groups. Such a molecule cannot be self-ligated by DNA ligase (which needs the phosphates). However, it's still possible to join the vector to the insert (which hasn't been phosphatase treated). There will still be a nick at each vector-insert junction, but that gets taken care of in the E. coli cell. Alkaline phosphatases procedure will reduce the number of blue colonies.

What happens if you add only 1 primer instead of 2?

You wont amplify it (draw it out.. you'll realise why) 1 primer only makes a new copy, but that 1 primer wont bind to the new copy. And also you'll only have the tiny amount of DNA as a template, and that template might bind to the new product, so you wont have any template left available. Thats why you have excess primers I think?

How would you disrupt the bacterial cell wall? Why would you do this?

You would do this to get at the protein inside Many research labs use sonication (application of ultrasound) for this, but it is not suitable for a practical class. You will use a commercial solution called BugBuster® that is a mixture of detergents that perforate the cell wall without denaturing the proteins (which most detergents do). You will help the process along by adding the enzyme lysozyme, which as you know from IA Biology of Cells, breaks the peptidoglycan layer in bacterial cell walls. The final addition to the lysis buffer is the nuclease, DNase I. When the cell walls are disrupted and the contents of the cell are released the solution becomes very viscous due to the large amount of DNA present. DNase I cleaves the DNA into smaller pieces and prevents the high viscosity.

Making primers with restriction sites?

You would ensure that the PCR product contains DNA sequences at its ends that enable it to be cleaved by restriction endonucleases so that the amplified target sequence can be cloned into an appropriate vector. Any such extra sequences are added to the 5' ends of the primers so that they do not interfere with copying the target sequence (where nucleotides are added to the 3' end of the primer) As the extra sequences are included in the primers they will quickly be amplified even though they are not in the original target sequence template Also, some additional nucleotides( typically 2-6 nts) are also added to the primer sequence upstream of the restriction site, flanking it, to ensure that the restriction enzyme can bind efficiently. Brandon: So if you have 20+ nts in the primer that are complementary to the cDNA, the primer will still bind even though the restriction site sequence (the new part added to the primer) isnt in the cDNA. So thats how you can add restriction sites to the primer.

What is subcloning?

a technique used to move a particular DNA sequence from a parent vector to a destination vector.

What are spectrin repeats?

are found in several proteins involved in cytoskeletal structure. These include spectrin, alpha-actinin, dystrophin and more recently the plakin family. The spectrin repeat forms a three-helix bundle. These conform to the rules of the heptad repeat. Spectrin repeats give rise to linear proteins. This however may be due to sample bias in which linear and rigid structures are more amenable to crystallization. There are hints however, that some proteins harbouring spectrin repeats may also be flexible. This is most likely due to specifically evolved functional purposes.

Chromatin Packaging Histone octamer - the protein core of the nucleosome

b) H2A-H2B dimer H3-H4 tetramer

What is cDNA?

cDNA is DNA synthesized from single stranded RNA (eg mRNA or miRNA) cDNA is generated from reverse transcriptase Brandon: I think its presented in 5' to 3' direction?

What is southern blot?

detection of a specific DNA sequence in DNA samples. transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

What is directional cloning?

directional cloning generally refers to the ligation of an insert into a vector in a known orientation. The most commonly used method I've dealt with is to cut the 5' and 3' sites on the vector and insert with different restriction enzymes that yield different sticky ends. This ensures that when you ligate insert into vector, the different identity of the 5' and 3' sticky ends ensures only 1 possible orientation for the insert to effectively anneal to the vector.

Tell me about University of California Santa Cruz (UCSC) Genome Browser

e.g. screenshot of UCSC Genome Browser showing MYL1 (myosin light chain) gene Mapping of transcripts to genome shows two TSS, separated by > 10 kb The University of California Santa Cruz (UCSC) Genome Browser is one of the two major web resources for viewing genomic information (the other is ENSEMBL). The view below shows 25 kb of human chromosome 2 encompassing the MYL1 gene. At this scale, you cannot directly view the nucleotide sequence, although it is trivial to zoom in on regions of interest (e.g. promoters, exons) to see the sequence. cDNA/mRNA sequences are mapped onto the genome; the thick parts of these lines represent sequences present in the mRNA (exons). The thin lines with chevrons are sequences in the genome that are not present in the mRNA i.e. introns. We can see that the MYL1 gene has two transcription start sites, and that exons 3 and 4 are alternatively spliced (see J Mata lectures) This URL will take you to the MYL1 gene on the UCSC human genome browser - you can use the "zoom in", "zoom out" and "move" buttons to take a closer look (purely optional exercise for those who are interested) http://genome-euro.ucsc.edu/cgi-bin/hgTracks? db=hg19&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=defau lt&virtMode=0&nonVirtPosition=&position=chr2%3A211154868%2D211179895&hgsi d=217981594_dBl68wFIQUA4UjEJHoMuXLOaVc8p

What is electroporation?

electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell Brandon: "poration" probs means introduce pores... clue it increases membrane permeability.

forward primer which one?

forward primer matches the coding strand, but attaches to template strand

Tell me about adding restriction enzyme sites to primers when cloning PCR products into a vector

gives rise to PCR products with sticky ends that are cloned efficiently. If different restriction sites are used, cloning will be directional. Example of PCR primers incorporating restriction sites on picture. Add restriction sites at the 5' end of each primer, need additional flanking nucleotides maximise the digestion efficiency. The number of additional nucleotides included depends on the restriction enzyme used. In the example above, it is necessary to include 3 residues adjacent to the HindIII site, but only 1 next to the EcoRI site. After the PCR reaction the products are digested with the restriction enzymes, then ligated into a suitable vector (for example, see the BMB practical, week 1).

Thermococcus litoralis

has proofreading polymerases. These marine bacteria grow at higher temperatures than Thermus aquaticus. The polymerases are more thermostable than Taq enzyme. T. litoralis can grow at temperatures up to 98 C and the enzyme retains over 90% of its activity after incubation for 1hour at 95 C.

What is heterologous expression?

heterologous expression means that a protein is experimentally put into a cell that does not normally make (i.e., express) that protein

Km

if you have an equilibrium, Km is a measure of binding

Tell me about Microinjection of DNA into the pronucleus of fertilized eggs at single cell stage when making transgenic animals to analyze the function of your protein

in this case, the DNA randomly integrates into genome. This means DNA may be inserted into a site on the host DNA that will enable its expression. Microinjection of DNA is used to insert new genetic information into the mouse genome or to overexpress endogenous genes. Experiment Example for Essay: In one of the earliest and most striking examples (1981), a RAT growth hormone gene was fused to a MOUSE metallothionein promoter and injected into fertilized eggs. 21 mice developed, 7 carried the growth hormone gene (randomly integrates) and 6 grew much larger than their littermates. Those with the highest copy number grew to double normal size and had 100-800x the normal concentration of growth hormone in the serum. Transgenic animals can now be produced for many different species, including farm animals.

Tell me about kcat

kcat is the normalized number for Vmax. kcat = Vmax/[E]. Its a first order rate constant that is concentration independent. Characterises the enzyme efficiency and enzyme saturation.

Chromatin Packaging Micrococcal nuclease releases nucleosomes

light digestion with nuclease more extensive digestion gel electrophoresis bp 800, 600, 400, 200

log distance vs BP OR log bp vs distance?

log bp vs distance how you remember this is that base pairs has a much higher variation, so this is the one logged

Give 3 examples of GFP variants

mCherry, Emerald, Venus

Primer design... You need to have some sequence information before you do PCR.... knowing a bit of the amino acid sequence of a protein encoded by the DNA..... How do you design a primer when you are uncertain about the sequence because you only have the protein sequence?

methionine and tryptophan have only 1 coding sequence, so for these you can be certain. Many organisms use some codons for a given amino acid in preference to others, so could guess codon. You can also use nucleotides such as inosine, that have broader pairing capabilities. Incorporating a mixed site at a position of uncertainty means we can be confident the correct nucleotide sequence will be present.The only thing is more likely to anneal to the wrong site.

Size exclusion chromatography?

method in which proteins in solution are separated by their size, and in some cases molecular weight chromatography column is packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or BioGel P). The pore sizes of these beads are used to estimate the dimensions of macromolecules

what is magnesium needed for in PCR?

needed as a cofactor for taq polymerase, Taq polymerase is a magnesium-dependent enzyme and determining the optimum concentration to use is critical to the success of the PCR reaction. Primers which bind to incorrect template sites are stabilized in the presence of excessive magnesium concentrations and so results in decreased specificity of the reaction. Excessive magnesium concentrations also stabilize double stranded DNA and prevent complete denaturation of the DNA during PCR reducing the product yield. Inadequate thawing of MgCl2 may result in the formation of concentration gradients within the magnesium chloride solution supplied with the DNA polymerase and also contributes to many failed experiments.

Why is it important that the gel system used for EMSA is non-denaturing sometimes....

otherwise the protein-DNA complex will not stay together while the gel is run. Thus SDS will not be included in the polyacrylamide gel or in the buffers this time. EMSA stands for electrophoretic mobility shift assay

Tell me about Piwi

regulatory proteins responsible for stem cell and germ cell differentiation Piwi proteins are highly conserved RNA-binding proteins and are present in both plants and animals. Piwi proteins belong to the Argonaute/Piwi family and have been classified as nuclear proteins. Studies on Drosophila have also indicated that Piwi proteins have slicer activity conferred by the presence of the Piwi domain. In addition, Piwi associates with Heterochromatin protein 1, an epigenetic modifier, and piRNA-complementary sequences. These are indications of the role Piwi plays in epigenetic regulation. Piwi proteins are also thought to control the biogenesis of piRNA as many Piwi-like proteins contain slicer activity which would allow Piwi proteins to process precursor piRNA into mature piRNA.

reverse primer which one?

reverse primer complementary to coding strand, 3' end of coding strand.

PCR the basics?

synthesize two oligonucleotide primers, one for the left hand end to direct DNA synthesis to the right hand end (5' to 3') and one for the right hand end to direct to the left hand end (5' to 3'). Then get the DNA, primers in large molar excess, thermostable polymerase and dNTPs and heat to melt DNA, then cool to allow primers to anneal. Synthesise DNA. repeat this cycle. Primers should be in large excess... why? I guess so that all the DNA present in sample has a primer attached, allows amplification to be rapid. Typical cycles in a PCR reaction would be: 1. Heat to 94 ⁰C to denature DNA 2. Lower temperature to somewhere between 40-70 ⁰C (often, 50-55 ⁰C) for annealing 3. Raise to 72 ⁰C for DNA synthesis (an optimum temperature for many thermostable polymerases) 4. Repeat steps 1-3 about 30 times 5. Cool to 4 ⁰C Why do you cool to 4⁰C at the very end? BL: Maybe its because once you have finished your PCR reaction and have your product, you want to prevent any degradation/depurination so you lower the temperature. Also kinda stops the PCR reaction if you lower temperature - slows Taq.

What is the phase problem?

the phase problem is the problem of loss of information concerning the phase that can occur when making a physical measurement. The name comes from the field of X-ray crystallography, where the phase problem has to be solved for the determination of a structure from diffraction data. The phase problem is also met in the fields of imaging and signal processing. Various approaches have been developed over the years that attempt to solve it.

What is a double crossover?

two separate crossing-over events occurring between chromatids. In a test cross involving three genes, progeny that have carried out this process can be identified and usually from the least frequent type of offspring.

What is a cap analog?

used for the synthesis of 5' capped RNA molecules in in vitro transcription reactions. These molecules are translated more efficiently in in vitro translation systems than uncapped mRNAs.

Prokaryotic Transcription Mechanism Identification and characterization of promoters Consensus Sequences

we will look at the experimental approaches that investigators can use to identify promoters. A number of these approaches can be more generally used to investigate protein-DNA (or protein-RNA) interactions Generating "consensus sequences" by aligning multiple sequences with respect to a common reference point (e.g. the TSS), is an obvious first step, especially with the wealth of sequence information now available (an in silico approach). Frequently, the degree of match to the consensus correlates with functional strength e.g. Lac-UV-5 is a strong promoter with a perfect consensus -10 box The sequence logo is a popular format for illustrating sequence conservation. The overall height at each position shows the degree of conservation, and the frequency with which each base occurs at that position. This logo was derived from the -10 boxes on the last slide.

How would you check the size of the PCR amplified DNA on a 0.8% agarose gel?

• Add 5 µl of the purified PCR product to a new microfuge tube. • Add 4 µl 6x Loading Buffer. This contains glycerol to make the sample dense and stop it diffusing out of the well into the buffer, and a blue dye (bromophenol blue) to help you judge how far the electrophoresis is going. • Add 11 µl of H2O to give a final volume of 20 µl. You should also have a tube containing 20 µl of the 100 bp DNA ladder (labelled M1), which contains multiple DNA fragments of known size. Loading the gel is the most difficult part of the whole practical - if in doubt about the loading, ask a demonstrator for help. It is best to use 2 adjacent tracks in the centre of the gel, rather than at the side. Draw up the contents of one tube into a Gilson tip. Without pressing down on the plunger, poke the tip underwater into a well in the gel (over one of the red lines). Do not jab the tip into the gel itself, keep it in the well. Then gently depress the plunger to deliver the sample slowly into the well. Don't worry if it won't all go in. Connect up the gel apparatus, with the negative terminal (black) at the end where you loaded the gel. The gel contains the same electrophoresis buffer that it is submerged in (10 mM Lithium acetate, 10 mM Boric acid, pH ~6.7). Switch on and run at ~150 V on constant voltage until the bromophenol blue dye has moved to the second set of red lines. This should take approximately 30 - 45 minutes.

Tell me about Fusion with Glutathione S-Transferase (GST)

• Addition of a 'GST-tag' (26 kDa) can also be useful for purification or other applications (E.g. Pull-down assays to look at protein-protein interactions) • Expression of the fusion protein is induced in E. coli, and crude lysates containing the fusion protein (and millions of others) are then prepared. • The resulting protein can be purified on a Glutathione sepharose column - and eluted with gluathione (competes for binding). Alternatively, the protein can be eluted from the column via protease cleavage if a site is present. In this case the GST part is removed. • The GST part can be cleaved after elution if necessary (when eluted with glutathione).

Tell me about Fusion with MalE maltose binding protein (MBP)

• Addition of a 'MBP-tag' (42.5 kDa) can be useful for increasing the solubility of a protein (In these systems, the protein of interest is often expressed as a MBP-fusion protein, preventing aggregation of the protein of interest. The mechanism by which MBP increases solubility is not well understood.), as well as for purification and other applications. • Expression of the fusion protein is induced in E. coli, and crude lysates containing the fusion protein (and millions of others) are then prepared. • The resulting protein can be purified on an Amylose column - and eluted with maltose (competes for binding). MBP is encoded by the malE gene of Escherichia coli.

How will selective media allow us to select plasmids with inserts?

• Cells which took up the insert only or no DNA will be killed by the ampicillin. • Cells which took up a plasmid with no DNA insert will survive and form a colony. The cells in the colony will have active ß-galactosidase, and the colony will be blue because of the Xgal. • Cells which took up a plasmid with an insert won't have active ß-galactosidase (as the gene was inactivated by the inserted DNA) and the colony will be white. Those are what we want.

Prokaryotic Transcription Regulation Positive regulation by CAP - 1. DNA binding

• Dimer of 22 kDa subunits. cAMP-bound form binds symmetric site upstream of RNAP • DNA binding via helix-turn-helix motif • 7aa "Positioning" or "stabilization" helix 2 makes non-specific contacts with backbone • 4 aa β turn • 9 aa "recognition helix" 3 docks into major groove. Sequence-specific recognition by H-bonding e.g. between Arg 180, Glu 181 and Arg 185 side-chains and base-pairs (direct and H2O mediated), and van der Waal's contacts • Helix-turn-helix motifs widely used for sequence specific binding by numerous dimeric bacterial DNA binding proteins (e.g. lac repressor, λ repressor and Cro) Typically recognize 2 "half-sites" separated by 1 helical turn - 34 Å No universal recognition code i.e. specific amino acid-base complementarity

Popular reporter genes include:

• Green fluorescent protein (GFP) and various other colours of fluorescent protein • Beta-glucuronidase (GUS) • Beta-galactosidase (X-gal staining) • Luciferase - renilla luciferase or firefly luciferase - have different substrates • Chloramphenicol acetyltransferase (CAT)

Tell me about RNAi generally

• RNAi is triggered by the presence of long dsRNA. • The dsRNA is processed into short interfering RNAs (siRNAs), which are key intermediates in the RNAi pathway. dsRNA processing into siRNAs is catalysed by an RNase III ribonuclease called 'Dicer'. o siRNAs have specific characteristics that are important for their recognition in the RNAi pathway - E.g. double stranded, 2 nucleotide overhangs at the 3' ends, 5' phosphate, ~21 nt in length. o siRNAs have one strand described as the 'guide' strand (the strand that will ultimately target the mRNA) and the 'passenger' strand (this strand will be destroyed). The choice of the guide strand depends largely on the stability of the 5' ends of the siRNA. • siRNAs assemble into an RNA induced silencing complex (RISC), where the 'guide strand' of the siRNA will ultimately be active for specific targeting of a mRNA. The essential protein in the complex is Argonaute 2 (Ago2), which has the 'slicer' activity that cuts the targeted mRNA. • siRNAs have perfect complementarity with their target mRNA, which is why they can target mRNAs so specifically. Essentially the duplex that forms between the mRNA and siRNA is a means of recruiting Ago2 for subsequent cleavage. • The mRNA target undergoes endonucleolytic cleavage by Ago2, which occurs at a very specific position - the cleavage position is located between nucleotide 10 and 11 when counting in an upstream direction from the target nucleotide paired to the 5'-most nucleotide of the guide siRNA • General cellular decay pathways subsequently degrade the remainder of the mRNA

Epitope-Tagged proteins

• Small tags (<20 aa) may also be fused to your protein - E.g. You might fuse a 'Flag' (8 amino acids), 'Myc' (10 amino acids) or 'HA' (9 amino acids) tag to your protein. • Epitope tag can be recognized by specific antibodies - E.g. immunofluorescence, immunoblots (Western blots), immunoprecipitations, etc o Useful to have an easily recognizable tag as antibodies are not available for all proteins, or that are good for all applications Example: An immunoblot (Western blot) was used to look at the distribution of HA-tagged phosphorylated-IRF3 (HA-p-IRF3) and Flag-tagged IRF3 (Flag-IRF3) in cytoplasmic (C) and nuclear (N) fractions isolated from HeLa cells. Lamin A/C was used as a control for the nuclear fraction and Tubulin was used as a control for the cytoplasmic fraction. Use of these controls confirms that the fractionation was relatively clean.