Molecular Bio Final (Stuff since Midterm 3). Study in conjunction with other stuff

RNA-seq

# of reads -- rna expression.

• If you know the sequence of a particular strand of DNA and want to determine whether proteins bind to it

(EMSA) - (lec 6, slide 19) two batches of DNA, one purified and isolated, and the other with cell extract (which contains DNA-binding protein) are fragmented and run in a gel. If there are proteins bound to the DNA, the fragments will not run as far down on the gel (they are heavier) *These fractions can be excised from the gel to isolate the DNA/protein complexes. This then also resolves which proteins are binding, but requires a step beyond traditional EMSA.

mRNA degradation

1) 3' end, shortening of poly A tail the exosome degrades it 2) 5' end, decapping (pyrophosphate linkage cleaved) xrn is the 5'3' exonucleus o This requires degradation from both the 5' and 3' ends of the transcript, to increase efficiency. The 5' end is always decapped and degraded by a 5' to 3' exonuclease, but the 3' end can be degraded in one of two ways: • Poly-A shortening - the poly-A tail is degraded first by deadenylase. Once this has proceeded to a critical point, recognition of A/U-rich elements (AREs) in the poly-A tail by AU-binding proteins (AUBPs) results in recruitment of the exosome, which continues degradation of the transcript from 3' to 5' • Endonucleolytic cleavage - rather than degrading the poly-A tail, specific sequences in the 3' UTR of the transcript signal for endonucleolytic cleavage, after which 3' to 5' degradation occurs. 3' end AU rich element=ARE AUBP binds to ARE, signals degradation from both ends.

Positive feedback loops

A is a transcription factor To stop the positive feedbck

Operator

A short regulatory section of DNA in the promoter that is recognized by a repressor protein, such as trp repressor

Trp attentuation

Attentuation Trp operon U rich seq.—intrinsic terminator -3/4 terminator Low tryptophan: Ribosome stalls—no trp trna to enter A site...so ribosome stalls. Doesn't get to region 2 Doesn't cover up the RNA 2/3 not a terminator (no nearby U-rich seq.) o Trp attenuation - trp synthesis is responsive to the amount of trp present in the environment. This is based on rate-limiting synthesis of the trpL (leader peptide) sequence. TrpL has two adjacent tryptophan residues near the beginning. Since trp is an uncommon amino acid, peptide formation is highly responsive to fluxes in trp levels. • The trpL gene has four distinct sequences, which can form stem loops (1/2, 2/3, or 3/4) that affect transcription, collectively serving as an effective regulatory mechanism: • High trp - trpL is translated without an issue, so the ribosome blocks 1 and 2 as it proceeds, allowing for a 3/4 loop, which functions as a transcription terminator. This prevents transcription of downstream enzymes for tryptophan biosynthesis. • Low trp - lack of trp causes stalling during trpL translation, so only sequence 1 is blocked. A 2/3 stem loop forms, preventing formation of the 3/4 terminator. Because of this, the 2/3 loop is recognized as an anti-terminator. RNAP can proceed and transcribe genes necessary for trp biosynthesis. • Extremely low trp or translation shutdown - if very little amino acid is present, two stem loops will form, 1/2 and 3/4. 1/2 exposes a stop codon early in the transcript, impeding translation altogether.

Genetic switch

Bacteriophage lambda

CAP is an ____, LAC is a ____

CAP-activator lac-repressor

Cassette switching

Cassette model of yeast switching Alpha or A type—both haploid Diploid- both alpha and A Dna recombination at MAT locus alpha info in A site and vice versa Double strand cut by HO endonuclease

Transcriptional activators are bipartite

Consisting of activation domain (AD) and binding domain (BD) which is what binds to the promoter directly

Chromatin remodeling and modifications

Covalent modification of histones Chromatin remodeling increases DNA accessiblity- facilitates binding of mediator, rna po., general transcription factors

Which base is the only dna that become methylated

Cytosine DNA methylation does not occur in yeast and flies

Helix turn helix

DNA binding motif two helices, with the recognition helix determining specificity by matching a unique amino acid sequence to a particular strip of bases in the DNA, and the second helix stabilizing the protein-DNA interaction. Usually HTHs function as homodimers, allowing for more specific binding interactions with greater affinity. This requires two site of recognition, and these are often palindromic (since it is recognized by a homodimer). helix-turn-helix motif of bacterial gene regulatory proteins is often embedded in different structural contexts, the helix-turn-helix motif of homeodomains is always surrounded by the same structure Homeodomains are one class of HTHs. Frequently these must interact with other DNA binding proteins, as they are not highly specific (many residues bind to the backbone of the DNA in addition specific bases). These can bind to both DNA and RNA.

Enhancers

Enhancers - genetic sequences that bind proteins to activate transcription. If they are far from the site of transcription, the large Mediator protein complex facilitates interaction of enhancer-bound proteins with RNAP.

Regulating the regulators

Eukaryotes regulators interact with many different ligands, proteins, and other factors, all of which contribute to their regulation through different mechanisms. You can find them all on this slide. The take-away point here is two-fold. o First, these are not mutually exclusive - you can have a transcription factor bound to an inhibitor protein, which when phosphorylated releases the transcription factor to enter the nucleus and stimulate protein synthesis of another gene regulatory agent. o Second, these occur under different time scales. For example, if you notice differences in activity within the span of seconds, that is likely due to some covalent interaction (i.e. protein phosphorylation or ligand binding). On the other hand, if the response only occurs after say 30 minutes, that is likely a more comprehensive process (i.e. de novo synthesis of proteins that results in the response).

T/F prokaryotes can have a downstream control element

F, but eukaryotes can have enhancers downstream

Cytosolic aconitase

Functions as enzyme-- determines amt of citrate in the cells Only works when bound to iron...senses iron levels No iron--> not active, but binds to two dif. mRNAs (ferritin and transferrin) Ferritin is iron storage protein binds in 5' utr, blocks translation... binds to 3' utr for gene for transferrin receptor...stabilizes it and its translated • Example (ferritin/transferrin) - Ferritin is an iron carrier, sequestering it when there is excess iron, whereas transferrin receptor is an iron scavenger, collecting iron from the cytosol when it runs low. Both ferritin and transferrin are regulated by the same protein, cytosolic aconitase, but in different ways. Aconitase has a binding site for iron, so it is responsible to cellular fluxes in iron levels. o With ferritin, aconitase binds near the 5' end of the transcript, preventing translation when iron is scarce. However, when iron is abundant, it binds to aconitase, releasing it and allowing for ferritin translation to sequester excess iron in the cell. o With transferrin, aconitase binds near the 3' end when iron is scarce. This masks a site for endonucleolytic cleavage, allowing for translation of transferring and scavenging of iron while it is scarce. However, when iron is abundant, it binds to aconitase, releasing it and exposing the cleavage site, causing mRNA degradation and impeding translation to prevent excess iron from being imported into the cell. Hairpin itself doesn't block the ribosome

Positive regulation in prokaryotes

General mechanism - binding of activator protein increases expression of a gene. Multiple possibilities (i.e. ligand binding removes activator protein to shut off expression, or ligand binding allows for activator to bind DNA and increase expression).

Activation and repression thru chromatin

HP1 (heterochromatin protein 1) binds H3K9me and forms heterochromatin

Helix-loop-helix

Helix-loop-helix - these usually dimerize as well, facilitated by a smaller helix, while the larger helix has a basic amino acid stretch that bolsters its affinity for interaction with DNA c-Myc

Repressive DNA methylation and histone modification (methylation) are linked

MBD (methyl binding domain) attracts HDAC, which deacetylates Also MBD attracts SET to methylate histones (repressive) Or other direction: Chromodomain protein recognizing methylated residue in proteins (H3K9 methylation) --recruit DNMT (methyltransferase)

Readout- the proteome

Molecular weight vs. isoelectric point --red protein spots-- common to both samples, blue, unique to individual sample

Retanoic acid receptor

No retinoids- it's a repressor RAR (retainoic acid response) LBD (ligand binding domain) LBD interacts w corepressor, interacts with H-DAC Transcription off under this condition In presence of retinoids Binds coactivator, recruits a HAT (acetylates the nucleosomes, facilitates activation) Mediator

B dna

Normal dna right handed 10-10.5 nucleotides per turn

Pyruvate kinase

Pkm2 slower than pkm1. Makes less atp Instead, there's accumulation of all these intermediates (helps make amino acid, phospholipids, nucleotides) • Pyruvate kinase performs the last step in glycolysis, producing pyruvate. Normal adult cells express PKM1, whereas tumors usually express PKM2. PKM2 is a less efficient form of the enzyme, which results in 'backing up' of glycolysis, and shuttling of glycolytic intermediates towards other pathways for biomass generation (AA, phospholipids, which is of more import to a cancer cell as that fuels proliferation. • This is an example of mutually exclusive exon inclusion. Normally, exon 9 is incorporated into PKM1. However, in cancer cells hnRNPs block the exon 9 splice site, resulting in incorporation of exon 10 instead of exon 9 into PKM2.

Production of ciRNAs

Production of ciRNAs. Circular intronic RNAs are produced by eukaryotic spliceosome-mediated splicing. The lariat intron generated from the splicing reaction evades normal debranching and degradation, and instead the 3′ "tail" downstream from the branchpoint is trimmed resulting in a stable ciRNA.

Antitermination in bacteriophage lambda

Protein N -transcribed downstream of cI N protein binds to the polymerase, changes its structure Prevents rna pol from termination (antitermination) o N protein (anti-termination) - normally bound to RNAP, nusA recognizes transcription terminator stem loops during intrinsic termination. However, N is able to bind to nusA that co-opts this affinity, such that N+nusA instead prefers binding to a specific nut sequence. When nut is transcribed, it binds to N+nusA, preventing nusA interaction with transcription terminators - resulting in a 'juggernaut' RNAP. Nut site (n utilization) Rouge terminator, ends t When sufficient N protein, works together with cellular protein...makes a complex N + NusA JUST GOES ******* rogue!!!! ignores the terminator ANTITERMINATION

Chromatin alteration

Proteins bound to enhancer regions can recruit different epigenetic factors, such as histone acetylases or chromatin remodeling complexes to increase transcription. Alternatively, gene expression could be suppressed by recruiting histone deacetylases, methyltransferases, repressive chromatin remodelers, etc.

U insertion

Puts Us on guide rnas (tutase)---addition of oligoU (NOT THE U's added itself!!!!) Blue part of guide base pairs to the mRNA Uridines also uses wobble to bind with the mrna Endonucleolytic cleavage at mismatch addition More uridine insertion via tutase again...just a couple uridines ligase Deletion Exonuclease removes uridine residues Ligase

RITS???

RNA-induced transcriptional silencing (RITS) is a form of RNA interference by which short RNA molecules - such as small interfering RNA (siRNA) - trigger the downregulation of transcription of a particular gene or genomic region. This is usually accomplished by posttranslational modification of histone tails (e.g. methylation of lysine 9 of histone H3) which target the genomic region for heterochromatin formation. The protein complex that binds to siRNAs and interacts with the methylated lysine 9 residue of histones H3 is the RITS complex.

Splicing is controlled positively and negatively--what kind of proteins?

Serine arginine--SR

Synergistic activation

Since eukaryotes often have multiple factors coordinately regulating a gene, they can act together to drastically increase the efficiency of transcription.

Malat1

Sr serine arginine proteins (rnabp—bind during splicing, activators of splicing) When malat1 is expressed at high levels...dec. of alternative splicing localizes to nuclear speckles, hotbeds of various proteins related to splicing, and sequesters SR proteins away from active transcription sites. This results in a decrease in alternative splicing in certain areas (in particular, MALAT1 is implicated in the aberrant splicing of several factors related to cancer metastasis).

Operon

The arrangement of adjacent genes coded as a single mRNA from a single promoter

HTH symmetric dimers

The group of helix-turn-helix proteins shown in Figure 7-l l demonstrates a common feature of many sequence-specific DNA-binding proteins. They bind as symmetric dimers to DNA sequences that are composed of two very similar "half-sites," which are also arranged symmetrically (Figure z-r2).This arrangement allows each protein monomer to make a nearly identical set of contacts and enormously increases the binding affinity: as a first approximation, doubling the number of contacts doubles the free energy of the interaction and thereby squares the affinity constant.

Coactivators and co-repressors

This is a concept in both prokaryotes and eukaryotes, namely that additional factors interact with regulatory proteins to promote or repress gene expression. While in prokaryotes these are often small molecules and fairly straightforward (i.e. trp binding to trp repressor impedes transcription), it is more complex in eukaryotes. For example, different combinations of the same group of proteins may recruit either co-repressors or co-activators for different genes, contextually defining the effect on expression rather than as an innate function of the proteins.

Zinc finger

Zinc finger - Zn ion coordinates the amino acid sequence into a conformation that facilitates DNA binding and recognition. Multiple zinc finger modules can be linked together in the same protein, allowing for greater specificity for a DNA sequence. For example TFIIIA has nine zinc fingers. alpha helix and beta sheet connected by zinc H23, H19 on alpha helix C3, C6 on beta sheet aka C2-H2 zinc finger (2 cysteines, two histidines complexing w the zinc) TFIIIA—the first Zn finger protein--- 9 zinc fingers...and binds both DNA and RNA they are cys2-his2 zinc fingers (each approximately 30 amino acids in size) arranged consecutively...1-3 more dna, 4-6 more rna

Zipcodes

Zipcodes—helps target where rna resides in the cell. (helps for optimizing synthesis of certain structures) very important in neurons and stuff. o The 3' UTR of a transcript often contains signals to localize the mRNA to a certain location to be translated, so that the protein product is close to its area of function. The transcripts can be moved to the appropriate place through a number of different vehicles: 1) transport on the cytoskeleton, 2) random diffusion and sequestration by proteins that recognize the localization signal, and 3) vulnerability to degradation except for those areas in which proteins locally bind and provide protection.

DNA looping

additional nearby lac operators the lac can bind (single lacR can bind 2 lacO simulatanously--> more repression)

Leucine zipper

bZip Two alpha-helices with leucines every seven residues- the coiled-coil o Leucine zipper (bZIP) - forms a 'zipper' like structure with a stretch of basic amino acids at the lower end of the bZIP, which interacts with the negatively charged phosphate backbone of the DNA to stabilize the complex. bZIPs are often found in heterodimers, which facilitates greater diversity of sequences that can be recognized, as well as greater specificity. Note, if homodimers, the sequences each alpha helix binds to is identical, however not the case with heterodimers (recognizes hybrid dna seq.) AP1, a c-Fos/c-Jun heterodimer

Major and minor groove

base-pair specific contacts in major groove H-bond acceptors and donors are recognized...as well as H atoms and methyl groups. The double helix structure of DNA results in two kinds of grooves - major and minor. Of these two, the major groove usually interacts with DNA-binding proteins (i.e. transcription factors) because it has more varied array of atoms available for hydrogen bonding interactions. This allows for greater combinatorial diversity in the recognition patterns of DNA-binding proteins, contributing to specificity of gene-protein interactions, which is critical.

IRES

binds eiF4g independent of cap bypasses cap structure multiple stem loop Normally in eukaryotes, the 5' cap and the proteins that bind it direct recognition for translation. In certain cases, however, tertiary structures arise from mRNA stretches labeled internal ribosomal entry sites (IRESs) that allow direct eIF4G/PBP binding and initiation of translation at that site, without the 5' cap or eIF4E recognition

Yeast mating type locus

diploid form MATa1 and MATα2 together turn off genes

Regulator protein structure

eukaryotic since regulator proteins in eukaryotes interact w other proteins, usually have 2 domains DNA-binding domain Activation domain

Trp receptor

example of neg. regulation in prokaryotes - when tryptophan (trp) is abundant, trp binds to the trp repressor, which shuts down genes involved in the biosynthesis of tryptophan. When trp is scarce, it is less likely to bind trp, and the trp repressor may dissociate from DNA. This is because of a conformation change associated with trp binding, which is necessary for it to bind DNA.

TF frequently bind as dimers to... and cooperatively to...

increase specificity increase affinity

XIST rna

lncRNA o XIST - (x-inactive specific transcript) regulates Barr body formation. Women have a mosaic pattern of gene expression because only one X chromosome, chosen randomly in each cell, is transcribed. There is an inactivation process that prevents transcription from the homologous chromosome. This process is regulated by XIST, which upon being transcribed, binds to the X-inactivation center (XIC) and coats the length of the chromosome. XIST subsequently recruits heterochromatin promoting epigenetic regulators (HDACs,H3K9 HMTs, DNMTs, HP1, polycomb) that further suppress transcription on one of the X-chromosomes. Xist transcribed from region of x chromosomes (x inactivation center) Responsible for shutting down entire x chromosome Xist rna does not escape and get to the other x chromosome (therefore only silences one) Spreads on the chromosome...induces heterochromatin like structure on the entire chromosome. Binding of proteins Hdacs, dnmt

Maintenance methylases

methylation is repressive

If you know the protein, but not the genes with which it interacts, and you want to identify the gene sequences it actually binds to in cells

o Chromatin Immunoprecipitation (ChIP) - cells are first treated with formaldehyde to cross-link and stabilize any protein-DNA interactions. The DNA is then extracted and sheared by sonication or nucleases. Bead-bound antibodies specific for the protein(s) of interest are then introduced and separated for further analysis. The protein-DNA crosslinks are reversed, and Proteinase K is introduced to degrade the protein, resulting only in the DNA fragments which were protein-bound. These fragments are then sequenced and mapped to a reference genome to determine the sites of protein binding.

• If you know the sequence of a particular strand of DNA and want to determine what proteins bind to it

o DNA affinity purification - total proteins extracted from cell lysate are run through a column containing various DNA sequences, allowing for isolation of DNA-binding proteins generally. Then, a second column with only the DNA sequence of interest is used, with a medium salt elution of non-specifically binding proteins, and a high salt wash eluting a protein with high affinity to the specific DNA sequence.

If you know the protein and the gene to which it binds, and want to identify the specific bases with which the protein interacts DNA footprinting

o DNA footprinting - the specific DNA sequence is amplified and labeled, exposed to the protein of interest, and cleaved (usually chemically) randomly. The exposure is done in such a way as to cut only once, producing a series of fragments of different lengths spanning each base. These are then run on a gel. The issing bands correspond to those sequences where the protein prevented cleavage of the strand, defining the recognition sequence the cleaving agent--small iron containing organic molecule that normally cuts at every phosphodiester bond w nearly equal frequency

Negative regulation in prokaryotes

o General mechanism - binding of repressor protein decreases expression of a gene. Multiple possibilities (i.e. ligand binding releases repressor to allow expression, or ligand binding allows for repressor to bind DNA and suppress expression).

Pos/neg. regulation in eukaryotes, general mechanism

o General mechanism - the concept is a bit more complicated here, as proteins themselves can have different activating or repressing functions depending on their interactions with their DNA sequences and other proteins. Also, the regulation is further complicated because nucleosome winding of DNA makes it possible for very distant regions to interact with each other in an enhancing or suppressing capacity, whereas interactions occur over much smaller distances, in general, in prokaryotes.

Production of sirnas and mirnas

o Producing siRNA/miRNA - a primary microRNA (pri-miRNA) is transcribed, consisting of a transcript with a stem loop. This is then processed by Drosha (part of the Microprocessor) to isolate just the stem loop (pre-miRNA). The pre-miRNA is then exported from the nucleus and further processed in the cytosol by Dicer to cleave off the loop (at the top of the stem loop, instead of base), producing a miRNA/siRNA duplex. The antisense strand is then incorporated into the RNA-induced silencing complex (RISC)—strands taken apart, which directs it to the appropriate transcript, to which it binds - now, it can have one of two effects that categorize it:---will then bind to mRNAs and exert regulatory function

• If you know the protein but not the gene with which it interacts, and you want to identify the nucleotide sequence it binds with the highest affinity

o SELEX - a combinatorial library of random ssDNA/RNA sequences of a particular length is formed and presented to a protein of interest. The protein is then pulled down and degraded, and any bound ssDNA/RNA sequenced. This sequence can then be used as a template for site-directed mutation to generate permutations that may exhibit more avidity than the original sequence. Over many iterations, one can find a single stranded sequence capable of binding with high affinity and specifically to the protein. o This can be extended further to try and identify possible genes with which the protein might interact, by mapping the optimal sequence onto a reference genome. Remember though, ChIP directly addresses the question of which genes a protein interacts with, whereas SELEX really only tells you the sequence of optimal affinity.

Lac operon

o What factors go into regulation? This controls expression of lacZ, which codes for β-galactosidase, the enzyme that breaks down lactose into glucose. This is only advantageous to the organism under two conditions: 1) glucose is not present, as glucose is a more direct energy/carbon source, 2) lactose is present, as otherwise the enzyme has no substrate and the organism uselessly expends both energy and biomass, which is particularly damning in a situation where it is glucose deficient and already struggling o Thus, the lac operon must be coordinately regulated by protein factors that can both sense glucose absence and lactose presence • Catabolite Activator Protein (CAP)--IT"S AN ACTIVATOR - activates expression when it binds cAMP, which is elevated when there is no glucose. • Lac repressor - normally represses expression, but detaches when it binds lactose, allowing for lacZ expression. o Neither CAP nor Lac repressor activity is sufficient alone to allow expression, but when CAP is bound and lactose-bound Lac repressor detaches, expression of lacZ is allowed.)

ceRNAs

o ceRNA - (competing endogenous RNA) regulates the rate of degradation of mRNAs that have been produced. ceRNAs serve as molecular sponges for miRNAs. Normally miRNAs can degrade their cognate mRNA transcripts, but ceRNAs compete with mRNA for miRNA binding. ceRNAs, then, result in increased translation of the mRNA which they are out competing. Some ceRNAs are circular. These can arise from "back-splicing," or in some cases stable intron lariats where the 3' extension is removed by the exosome. Bind to microRNAs, not to proteins Low ceRNA free miRNAs silence expression High ceRNA less free miRNAs activates expression (miRNAs not binding the mRNA as well)

Shutting down cap dpeendent translation

o eIF4E binds the 5' cap. Together with eIF4G, PBP and other factors, it allows for translation of mRNA transcripts that have been capped. Then, this protein-mRNA complex is shuttled to the small ribosomal subunit to initiate translation with eIF2-GTP, as described above. eIF2 and eIF-2B are critically involved in regulation of the global translation processes. • eIF-2B is a guanine nucleotide exchange factor that is able to transfer GTP to the GDP-bound eIF2 (which is inactive) to replenish the active GTP-bound version. During certain cellular stresses, GDP-bound eIF-2 is phosphorylated on its alpha subunit. This phosphorylated GDP-bound eIF-2 has high affinity for eIF-2B, sequestering it and inhibiting its GTP exchange activity. This results in a depletion of eIF-2 in its active form, shutting down cap-dependent translation. Needs help of eiF2B (guanine nucleotide exchange factor) eiF2 hydrolyzes GTP and leaves GDP eiF2 facilitates binding of mettRNA and to keep it in place and also to prevent association of 40s subunit too before AUG GDP bound EiF2 needs to be recycled eiF2B binds to inactivate complex, facilitates release of GDP, allowing it to bind another GTP, eiF2b releases, can cycle multiple times. Phosphorylation can affect this process -dampens the translation -activated by stress -phosphorylation makes the complex not functional, and eif2b can't facilitate release of gdp, and the complex just clumps together, slowing down translation.

Only difference btw pos and neg. regulation

pos has activator protein neg has repressor protein

Lambda repressor

prokaryotes Example (lambda repressor) - this example can be either positive or negative depending on it's the location of operator (binding sequence) to the promoter. If there is sufficient distance, the repressor is able to bind without impeding RNAP loading and activate gene expression. However, for other genes, the operator is closer to the promoter, so binding of the lambda repressor prevents RNAP loading, decreasing gene expression.

Nitrogen transport

prokaryotes Example (nitrogen transport) - when an alternate sigma factor (bacteria and viruses contain many of these, with different promoter specificities) binds to a gene for nitrogen transport, it is able to interact with a protein (NtrC) bound to a distant enhancer via DNA looping (as with the lac operon, described later) to activate expression of nitrogen transport genes. The looping around displaces the sigma factor. sigma factor54 itself is not strong enough to activate transcription

CpG islands

regular C--> U (deamination) -easy to detect 5-methyl C-> T (deamination) -hard to detect Intact CGs that haven't been methylated are in these CG islands. Promoters are enriched in CG islands Housekeeping genes. CG are methylation sites (usually repressive)...why constitutively active CG's are pretty rare Resistant to methylation

HIV tat

tat protein (HIV) - recruits P-TEFb to phosphorylate serine 2 of the RNAP CTD domain, which results in unpausing and elongation of HIV genes.

Human b-globin the figure???

there is massive overproduction of hemoglobin in certain cells - how does this occur? In this case, there is a locus control region far upstream from the promoter of the globin gene that serves two roles: 1) recruitment of histone remodeling complexes, histone acetylases, etc. to activate transcription, and 2) serves as a barrier sequence to prevent spread of heterochromatin to the gene locus, which often occurs shortly after transcription during a 'refractory' (my own terminology) period or more stochastically throughout the genome. This dual function contributes to the high rates of transcription of this gene. Control region not really an enhancer (too close to promoter) Locus control region prevents heterochromatin from forming, keeps it open- so transcription factors can come thru

Riboswitch

this generally refers to an mRNA that adopts a tertiary structure to bind a small molecule, and upon doing so, experiences a conformation change that alters its transcription. For example, a transcribed sequence upstream of genes coding for enzymes involved in purine biosynthesis has a structure that allows it to bind guanine. When guanine is not bound, purine biosynthesis genes are left on, but upon guanine binding, a transcription terminator loop is formed, turning off expression of those genes due to purine abundance. RNA binding to ligand itself Ppp (prokaryotes) It binds guanine, causes it to fold in a different way

Phase variation

this regulates two genes that code for flagella in opposite orientation (H2 and H1). Both H2 and H1 have separate promoters, but the sequence of genes is arranged linearly such that H2 is transcribed first, followed by a repressor that blocks H1, and H1. So long as the system is intact, this results in exclusively H2 production, as the repressor blocks all H1 activity. In rare instances, however, an inversion of the H2/repressor promoter can occur, preventing their expression, freeing the H1 promoter from repression and allowing for H1 expression. o This occurs very rarely (1 in 100,000 events), but serves an important evolutionary mechanism. Cells often produce antibodies to flagella to terminate bacteria, however, these antibodies are specific to the orientation of the flagella, and therefore do not recognize the opposite orientation given by H1, allowing those bacteria to survive.

Gene control region

whole expanse of dna involved in regulating and initiating transcription of a gene, including promoter, and all regulatory sequences to which gene regulatory proteins bind to control rate of assembly at promoter

Alternative splicing

• Cassette exon - exon is either included or excluded • Mutually exclusive exons - either one exon is included or the other, but not both • Intron retention - the intron is either included or taken out • Alternative 5' or 3' splice sites -can choose between different sets of splice sites, which results in inclusion or exclusion of a small set of nucleotides. The splice site can be internal as well (i.e. within an exon). • Alternative promoters - transcription can begin at different sites. If there is a downstream promoter, that will be spliced.

Eve in drosophilia

• Eve in Drosophila - the eve gene is expressed in a specific spatial distribution, actively expressed in certain regions and repressed in others. How does this regulation occur? This process is coordinately regulated by four factors: Bicoid and Hunchback (activating); and Kruppel and Giant (repressing). o Both Bicoid and Hunchback are required for activation, but either Kruppel or Giant is sufficient for repression (reflecting the general rule that a lack of expression is preferable to a gene being constitutively active). Thus, the relative levels of these factors determine where the stripe (indicating eve expression) is formed (Bicoid and Hunchback present, neither Kruppel nor Giant present). o Regulation is a result of competition for overlapping recognition sequences on the gene, such that a repressor prevents the binding of an activator to the same sequence. This is a common regulatory mechanism.

Membrane bound v. secreted antibody alternative polyadenylation...class switching

• Membrane-bound vs secreted antibody - stop codons are sometimes found in introns. In this case, splicing of the intron between two exons results in translation of a hydrophobic stretch coded by the second exon (allowing for membrane localization). On the other hand, if the intron is not removed, the second exon is never translated. The part of the intron preceding the stop codon codes for a hydrophilic stretch (allowing for secretion). • IgM (class switching) - B cells function as immune sensors, with a membrane-bound antibody that can bind antigens. When stimulated, these are then turned into plasma cells, which produce vast amounts of secreted antibodies. This transition occurs, in part, due to changes in alternate polyadenylation. IgM codes for the heavy chain subunit of the antibody, with part of its sequence responsible for embedding the antibody within the membrane. There are two sites for binding of the cleavage factor CstF, with different affinities. The higher affinity site is farther down on the sequence, resulting in default production of the membrane-bound antibody. However, when concentration of CstF increases, a sequence farther upstream (in the intron region) with lower affinity can be bound, resulting in a shorter transcript that translates to a secreted antibody.

Sex determining splicing cascade in drosophilia

• Sex determination splicing cascade in Drosophilia - this is an important and well-defined example of regulated alternative splicing that determines sex in Drosophilia • The primary signal for whether the fly develops as a male or female is the ratio of number of X chromosomes to the number of sets of autosomes (A). A fly with XY sex chromosomes (1 X) and two sets of autosomes has a ratio of 1:2 and will develop as males. A fly with XX (2X) and two sets of autosomes has a ratio of 2:2 and will develop as female. • Three crucial gene products in the cascade: Sxl, Tra, and Dsx. The MALE pathway is default, and the Sxl and Tra genes continually produce nonfunctional proteins due to the default splicing of their RNA transcripts. Dsx will produce a functioning, male-specific Dsx protein that represses genes specifying female characteristics. • The FEMALE pathway is activated when the X/A ratio is 1 (2X:2A), providing a signal that transiently activates a promoter in the Sxl gene and results in an alternatively spliced transcript. This transcript translates to a functional Sxl protein, which acts similarly to hnRNPs by repressing splice sites, or exerting negative control over RNA splicing. • Functional Sxl protein autoregulates its own RNA transcript splicing to result in more functional Sxl being translated. Sxl blocks the 3' and 5' splice sites of Sxl RNA exon 3, thus excluding that exon from the final transcript. In the default MALE pathway, inclusion of exon 3 results in a nonfunctional protein because Sxl exon 3 contains a premature stop codon (PTC). • Sxl also blocks a 3' splice site in Tra RNA, leading to a transcript that utilizes a downstream, alternate 3' splice site. This also splices out a PTC, leading to production of functional Tra protein. • Functional Tra exerts positive control over RNA splicing. It works with constitutively produced Tra2 to activate splice sites, similarly to SR proteins. Tra and Tra2 activate a splice site in Dsx, leading to the inclusion of an alternate exon 4. This female-specific spliced form of the Dsx transcript produces the female form of Dsx protein, which represses male differentiation genes.

Difference between siRNAs and miRNAs

• siRNA - perfectly complementary to the mRNA, resulting in degradation of the mRNA transcript itself • miRNA - imperfect complementary to the mRNA, resulting in a bulged helix that cannot be translated, but does not cause degradation of the transcript itself (argonaute does not actually cut, like it does for the siRNAs)--Translation inhibition ---labs still trying to figure out how. ---maybe prevents recognition of the cap structure, but targeted mRNAs can begin translation (find them associated with ribosomes) --but something funny during translation...recruit decapping enzymes as its going (maybe, maybe not)