Molecular Biology Exam 3

Different sigma subunits and their function

***70: housekeeping genes expressed in all growing cells 38: starvation/stationary growth phase 28: flagellar structure and movement 32: heat shock 24: extracytoplasmic stress 54: nitrogen uptake and metabolism

RNA polymerase uses synthesis mechanism analogous to that of DNA polymerase

-2 Mg ions fit into the active site of polymerase protein -incoming rNTP form watson-crick base pairs -interacts with 3' OH group allows for an Sn2 reaction -steric mechanisms are used to make sure the right rNTP is incorporated -Heavily favor Watson-Crick base pairs to be formed -one mg ion deprotonates water to form the nucleophile in the reaction to attract phosphodiester bond; makes better nucleophile -the second mg ion coordinates the removal of the pyrophosphate; coordinates the 3' OH of the leaving strand that acts as the leaving group -polymerase won't work without this

PCR process

-denature template strand with heat -anneal primers (more stable than rna); allow to add polymerase -elongate with DNA polymerase -heat and repeat the process as many times as needed product: defined by 5' ends of each primer

Splicing can allow diverse products to be created from a single gene

-gives us genome diversity and layers of regulation -because of the way splicing happens, you can make two different proteins from on egene alternative splicing allows specific exons to be selected for the mature transcript

How does antibiotic resistance genes allow us to select our recombinant plasmid?

-we start with a plasmid backbone that contains and antibiotic resistant gene -after we have inserted our gene of interest, we will transform the new recombinant molecule into the bacteria -when grown on an antibiotic plate, only the bacteria that took up the plasmid will grow

What is required to build a recombinant DNA molecule?

1. A vector that we can insert of gene into and then introduce into a cell for replication (usually into a bacterial plasmid with origin of replication) 2. A way to insert the gene of interest into the vector 3. A selectable marker that will let us select cells that take up the recombinant vector (determine which E coli cells have taken up recombinant plasmid and which haven't) 4. A second selectable marker that will let us select cells that actually contain our gene of interest (helps us sort out plasmids that contain gene of interest from those that don't)

Transcription occurs in distinct steps. Name them

1. Binding of RNA polymerase core to the DNA promoter (CLOSED complex) 2. Formation of transcription bubble (open complex) 3. Initiation 4. Elongation (promoter clearance) 5. termination and recycling Promoters direct the site of RNA polymerase binding and the initiation of transcription. Transcription always proceeds from 5' to 3' on the coding strand No RNA oligonucleotides necessary to initiate transcription (unlike DNA replication)

Differences in DNA replication and RNA synthesis

1. DNA = entire chromosome replicated exactly once; RNA = portions of chromosomes transcribed multiple times 2. During transcription, the nascent RNA strand is immediately displaced from template 3. Transcription is much lower fidelity than replication (1 mistake per 10,000 bases, as compared to 1 mistake per 10 million) -RNA can afford to be less accurate because it can produce something that is not permanent

Eukaryotic transcription by Pol II

1. Pol II recruited to DNA by transcription factors 2. Formation of transcription bubble 3. Phosphorylation of CTD during initiation 4. Elongation 5. Transcription terminates and CTD is dephosphorylated Polymerase binds to promoter region of gene, recruited there by a collection of transcription factors that all bind at promoter and for a complex called pre-initiation complex Once dna has melted, we have initiation complex\ Synthesizes bunch of short rna strands until clear promoter -After that it needs to clear to transcribe Tail is phosphorylated, which recruits enzymes that process rna to be transcribed

Process of capping

1. Removal of gamma phosphate by RNA triphosphatase 2. Addition of GMP by the guanyltransferase enzyme 3. Addition of methyl group to the purine and guanine of the new 5' base

how can we truly confirm the identity of a plasmid that we have constructed?

1. Restriction digest: does the digest create a restricition map consistent with the plasmid we are interested in? - we have a map at beginning with vector, insert and recombinant... so you know what the recombinant plasmid should look like 2. PCR: can we use PCR to amplify a sequence from the insert? if so it was inserted correctly 3. DNA sequencing: did the insert end up in the correct orientation?

major features of a recombinant DNA molecule

1. Two selectable markers 2. Origin of replication 3. Restriction sites flanking inserted gene 4. Primer binding sites for PCR or sequencing; used in sanger sequencing 5. Promoter for gene expression (optional)

similarities of DNA replication and RNA synthesis

1. both are the synthesis of a new nucleotide strand complementary to a DNA template 2. Both use a multi-subunit polymerase that adds free nucleotides to a growing strand 3. Synthesis in a 5' to 3' direction displaced RNA is not paired with its complementary strand

The spliceosome assembles on the intron to be spliced in a regular order. What is each step of the process?

1.) U1 binds at the 5' splice site (using base pairing between complementary sequences in the U1 RNA and intron RNA) while U2AF binds at branch point in intron 2.) U2AF will recruit U2 to bind to branch point using WC**** base pairing between RNAs- the base pairing between U2 RNA and intron RNA is not perfect, so branch point A bulges out!! -this is essential to allow it to present that 2' OH group found on bulge as an available group for first transesterification 3.) The tri-snRNP particle (consisting of U4, U5, & U6) arrives and replaces U1 while binding to U2, bringing the branch point and the 5' splice site together -this allows geometry (they are now close enough) for first transesterification reaction to occur 4.) The U4 snRNP leaves the complex, causing a rearrangement between the remaining U5, U6 and U2 snRNPs -these rearrangements allow the first transesterification reaction to happen, then the second happens next end product: joined exons and liberated lariat, makes 3 way junction at branch point

library of sequences

A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism and are stored as a "library"

Before elongation begins, the polymerase undergoes what process?

ABORTIVE INITIATION an early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the transcription complex leaves the promoter. RNA polymerase has to deal with several hurdles to get elongation started: 1.) No primer to provide the first 3'-OH group 2.) Sigma subunit is blocking the RNA exit channel of the polymerase 3.) Sigma is still tightly bound to the promoter region of the DNA

You have isolated an organism from the gut of a patient suffering a stomach ailment. You isolate an RNA sample from the organism, reverse transcribe it into cDNA, and sequence the cDNA. After aligning the result with a reference sequence, you find the following (solid = aligning sequence; dash = alignment gap) What explains this result? A. The sequenced cDNA is part of an operon B. The sequenced cDNA was synthesized from a spliced RNA C. The reference sequence contains transposable elements D. The reference sequence has undergone recombination

B. the sequenced cDNA was synthesized from a spliced RNA

What needs to happen to produce a mature mRNA molecule?

Bacteria: even as RNA is being produced, there are already ribosomes translating proteins- happens at same time -Very efficient -Transcript ready to go right away Eukaryotic transcript is not ready to go right away like bacteria is -Before ready to be translated: need 5' cap, intron spliced out, poly-A tail added to make mature mRNA****** -Once fully processed, can interact with nuclear pore complex, exported to cytoplasm and interact w ribosomes to be translated and go through degradation to control how much rna accumulates in the cytoplasm **capping, splicing and poly a tail addition happen at same time as transcription (coordinated by Pol II)

RNA processing happens at the same time as transcription, and it always proceeds in the same order (capping then splicing then polyadenylation). Why does capping always come first? A. The 3' end of the transcript always emerges from the polymerase first. B. The 5' end of the transcript always emerges from the polymerase first. C. The factors that cap/splice/polyadenylate are recruited by the tail in the order that they are used. Both A and C. Both B and C.

Both B and C

We have discussed several ways in which DNA and RNA synthesis are similar and different. One interesting difference is that several enzymes required for DNA replication are not required for transcription of RNA. Which of following enzymes are required for DNA replication, but not for RNA synthesis? A. Polymerase B. Primase C. Ligase Both A and B Both B and C

Both B and C Doesn't have a primer so do not need primase, only has one continuous strand as of now

How did Crick and Brenner demonstrate the no punctuation rule?

By using experiments in the T4 phage, which infects E coli 1. T4 phage viruses were mutagenized with proflavin which tends to cause insertion and deletion mutations 2. They looked for mutations that stopped the function of the rIIB gene, which causes the phage to lyse bacteria prematurely 3. They found that inserting or deleting a single nucleotide from rIIB caused a premature lysis phenotype - presumably due to a frameshift mutation 4. However, inserting and deleting a nucleotide did not cause a mutant phenotype - there was no frame shift in this case x

You have designed primers to amplify a DNA segment that you are studying. There are exactly 150 base pairs from the 3' end of one primer to the 3' end of the second primer. Each primer is 25 base pairs long. How long can you expect the PCR product of the reaction to be? 150 base pairs 175 base pairs 200 base pairs <150 base pairs >200 base pairs

C. 200 base pairs 5' end of each primer would define the length of the PCR

What does capping do?

Capping adds a methylated guanine base to the 5' end of the pre-mRNA transcript the capping enzyme (guanylyltransferase) is recruited to the c-terminal tail of Pol II 5' cap will be later used during export of the mature RNA from the nucleus and initiation of translation ***5' cap is important because it is used as a tag to let mRNA to be exported from the nucleus Guanylyltransferase is associated with the Pol II CTD to ensure that each mRNA is capped as it is transcribed Once the cap is complete, the guanylyltransferase dissociates and the cap-binding complex (CBC) binds

How do we get our plasmid into bacterial cells for growth?

E coli cells that are ampicillin sensitive Grown up in liquid culture until in log phase of growth -Are then harvested by centrifugation and then resuspended in cold calcium chloride solution Have plasmid dna added to cells (amp resistant gene in this) -Pores form periodically, but cold stabilizes it -Calcium shields negative charge on DNA can facilitate it to get inside**** ***Heat shock: creates thermal gradient that sweeps DNA into cells Return cells to growth media, then spread cells on agar plate that contain ampicillin Those that didn't take up the gene will be killed, and those that did will grow Incubated 24 hours, then colonies should be there

You have used PCR to amplify a potential disease-causing mutation from a sample of human genomic DNA. You have sequenced a portion of the PCR product and found that one position contains overlapping chromatogram peaks for A and G. What could explain this observation? A. A misincorporated base during the sequencing reaction. B. The person who provided the DNA sample possessed heterozygous sequence at that position C. The was a misincorporated nucleotide during the PCR amplification D. Either A or B E. Either B or C

E.

How does the Pol II core promoter sequence direct the location of initiation

Elements of the core promoter are bound by the general transcription factors and every promoter contains a subset of these promoter elements Additional regulatory regions (and their associated binidng proteins) provide additional layers of regulation the more complex promoter region gives us more layers of regulation of gene expression, allows us to have different types of cells within the body

Incorrect types of splicing

Exons can be skipped Pseudosplice sites: close to a sequence of splice site, sometimes the spliceosome confuses it; loses information

The mutations in the rIIB gene that Crick and Brenner were studying are called nonsense mutations, and they generally lead to a complete loss of gene function. Previous studies of the rIIB gene had identified transition and transversion mutations that lead to a partial loss of gene function. These are called missense mutations. Why do nonsense mutations generally cause a stronger phenotype than missense mutations? A. Nonsense mutations alter the function of a single codon B. Missense mutations alter the function of a single codon C. Nonsense mutations alter the function of every codon occurring after the mutation D. Missense mutations alter the function of every codon occurring after the mutation E. A and D F. B and C

F

Why is it that a transition mutation is more easily protected by wobble base-pairing than a tranversion mutation is? A. In a transition mutation, a purine base remains a purine and a pyrimidine base remains a pyrimidine B. In a transversion mutation, a purine base remains a purine and a pyrimidine base remains a pyrimidine C. In a transition mutation, a purine base becomes a pyrimidine and vice versa D. In a transversion mutation, a purine base becomes a pyrimidine and vice versa E. Both B and C F. Both A and D

F. If third position remains at least same type of base, a wobble base pair is more easily able to form If changed to different type of base, in trouble because a wobble base is unlikely to form a base of the same type

Structural changes in the holoenzyme convert the initiation complex from closed to open. How?

First binds: in a closed conformation/closed complex Sigma subunit melts the -10 element -10 is susceptible to melting bc it's mostly AT pairs so its easy to pull apart to single strands Now there is an open complex; complex theoretically able to perform trancription -Abortive initiation: transcribe a little, falls off, over and over for a series of them.... Eventually it makes progress and starts elongation and perform promoter clearance -Reasons for ^^ is part of sigma subunit is actually blocking the rna exit channel -One of things that has to happen is sigma has to change its conformation to unblock the rna exit channel Sigma is bound to promoter, rna polymerase is bound to sigma, but it has to leave promoter (dissociate from sigma) in order to get away to do its job Open and closed complex is talking about DNA melting state (after melted, single strand = open complex)

Group II self-splicing introns

Folds up into complicated hairpin One hairpin has A nucleotide bulging out of it The way they fold together leads to where bulging A nucleotide comes into close proximity of 5'splice site to allow for the two transesterification reactions **The intron is liberated in a lariat conformation** may have evolved first, autonomous self splicing sequences; genomes started evolving to have spliceosome components, so introns lost the ability to splice themselves

What does phosphorylation of the Pol II c-terminus allow?

It allows Pol II to escape the promoter During abortive initiation, the Pol II complex will produce short (<10 nt) transcripts before "escaping" the promoter Phosphorylation of the Tyr-Ser-Pro-Thr-Ser-Pro-Ser heptapeptide sequence in the Pol II c-terminus by TFIIH (and other kinases) allows to Pol II to escape the promoter and begin elongation of the transcript c terminal tail protein Tail makes tight interactions with all of them Good bc efficient Polymerase has to leave promoter, but its tail is in the way. Goes through series of abortive initiations Tail is still tied up so has to wait Allows polymerase to get on with polymeriztion

Some mutation can be suppressed by mutant tRNAs. Explain some types.

Missense: change in codon to change in amino acid (one amino acid is changed) -a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. It is a type of nonsynonymous substitution. Nonsense: have a transition or transversion that converts codon into a stop codon; terminates protein -sometimes early stop codon can be suppressed by mutant tRNA that has a compensatory mutation in its anticodon loop; adds tyrosine amino acid instead of stopping it -mostly of consequence experimentally -causes issues with real stop codons; if a lot of tRNA was floating around, a lot of real stop codons would be translated through; therefore, very rare bc if all stop codons were translated through, it would kill the organism; only viable when affecting rarely used stop codons

Is energy input required for splicing reaction?

No. Splicing chemistry is energetically neutral: two phosphodiester bonds are broken and two are created However, can use energy input for: -it to happen fast, putting energy in makes it happen quicker -energy can be used in order for the transesterification reaction to happen; the pieces need to be brought close together, and to make sure the relevant chemical groups find each other accurately

Why is the sequence required to direct splicing contained mostly within the intron?

Once splicing reaction happens, don't want any more splicing to occur The exons need to code for specific AAs to make proteins, if have splicing sequences, it will be constrained because they will always have that sequence in there

Prokaryotic initiation requires specific DNA elements called __________.

PROMOTERS Promoters provide landing spot for rna pol to come in and bind In bacteria, transcription begins with the core polymerase binding the promoter region of a gene view interactions be DNA and the sigma subunit Frequently consist of -10 and -35 regions named in reference to where transcription starts (at 90 degree arrow) given - number if upstream of site, separated by spacer -If look at regions, for a lot of diff e coli, there are spec sequences that are shared by many diff genes at the -10 and -35 regions Those sequences show up more often than not, -Consensus sequence: most commonly seen sequence --Strong consensus, probs some protein that binds and recognizes it Sigma subunit: binds to -10 and -35 and bind to rna pol in order to bring the rna pol to promoter **the -35 and -10 elements are not identical across different genes, but do have consensus sequences Bacteria have more than one subunit The most common sigma subunit in E. coli is σ70, which interacts with the -10 and -35 regions of the promoter

KINETIC PROOFREADING

Pol II mechanism for proofreading or avoiding errors How it works: -if the incorrect rNTP is added, progress of polymerase dramatically decreases -the slowing creates an opportunity for the SN2 reaction to run in reverse and liberate the wrong nucleotide back out Mechanism to fix errors: slow down to reverse (Following a misincorporation, the polymerase pauses in order to allow the reaction to run in reverse and release the incorrect NTP)

Nucleolytic proofreading

Pol II mechanism for proofreading or avoiding errors this happens with the wrong rNTP is incorporated and several rNTPs after that, so it cannot run in reverse Following a misincorporation, the polymerase backtracks and uses nuclease activity to chop off the segment of RNA containing the mistake uses hydrolysis reaction to chop off everything that happened after misincorporation neither this nor kinetic proofreading is as good as 3' to 5' exonuclease in DNA polymerase -allows mess ups at a much higher rate than DNA polymerase

Why is it important that promoters direct the site of RNA polymerase binding and initiation of transcription?

Promoters can direct pol to transcribe spec genes and make sure those genes are transcribed in its entirety

What does poly-A tail help with

Promotes stability of transcript Aids in translation and assembly of ribosomes (attracts ribosomes) Poly-a tail can be dozens to hundreds long Longer tails stick around longer than short, takes longer to degrade (allows it to figure out how long it sticks around by adding more or less A's)

Transversion

Purine replaced by pyrimidine C replaced by G or A So CG become GC or AT less common

Transition

Purine with a purine; CG switched with TA (C with T and G with A) these are most common Cytosine deaminated to become uracil, which would lead to an Adenine being incorporated, lead to eventually a CG base pair being replaced by TA Deamination of adenine, creates hypozanthine which can base pair with cytosine, over time AT base pair is replaced by GC can happen through spontaneous hydrolysis

What are the two mechanisms prokaryotic cells use to halt transcription?

RHO-DEPENDENT MECHANISM RHO-INDEPENDENT MECHANISM

Wobble bases

RNA capable of non-WC base pairs UGC in anticodon loop can bind to GCA codon through standard, but can also recognize GCG (G with U) -this allows one tRNA to recognize a wider variety of codons in mRNA, allowing the same AA to be incorporated across multiple codons Other way it works: Modified base: INOCINE -can form base pairs with C, U or A in the mRNA

rho-dependent mechanism

Rho is a complex that forms around the RNA transcript at rut sequences and then pulls the transcript out of the polymerase donut shape allows it to assemble around a growing RNA strand rho complex finds rut and binds to it, atp hydrolysis for energy processes along mrna molecule until reaches rna channel of polymerase and then pulls rna out of the polymerase Now polymerase doesn't have 3' OH group so cant continue synthesizing rna, falls off template and gets recycled ATP dependent complex

How is the accuracy of splicing ensured?

SR (serine-arginine-rich) proteins bind to exonic splicing enhancers to drive spliceosome assembly at intron-exon junctions this mechanism helps ensure that the spliceosome machinery will assemble at intron-exon boundaries SR proteins prevent spliceosome from binding in middle of exon and helps recruit U1 and U2 snRNPs that do need to bind to introns

Which sequencing method is appropriate for large scales and which for small scales?

Sanger: not super efficient, cost per base is pretty high, but does well on SMALL scale; don't know what primer to use; you have to start by knowing partial sequence to know the primer needed Pyro and illumina: cost per base is very low, but cannot be done on small scale; LARGE scale

You have designed primers to amplify the entirety of a large eukaryotic gene. You decided to carry out a standard PCR reaction with genomic DNA as template and an RT-PCR reaction with mRNA as template. Based on what you know about eukaryotic mRNA processing, which reaction would you predict will produce a longer PCR product. A. Standard PCR reaction B. RT-PCR reaction C. Both will produce the same size product D. Can't tell from the information given

Standard PCR reaction

T or F. There is no primer for transcription

TRUE RNA polymerase has to juggle the first two rNTPs to bind them together

What DNA polymerase is typically used in PCR?

Taq DNA polymerase -found in bacterium of a geyser in Yellowstone; extremely thermostable because since it lives in extremely hot conditions essential to PCR because it allows for DNA elongation to occur

How is the Pol II complex removed from DNA to complete termination?

The "torpedo" model of transcription termination in eukaryotes: 1.) An AAUAAA sequence is transcribed to signal polyadenylation (more on this process later) 2.) After the polyA mRNA is cleaved from the complex, the Xrn2 nuclease (5' 3') is recruited to the loose end of the RNA still associated with the polymerase 3.) When Xrn2 reaches the polymerase, it terminates transcription by pulling the transcript from the active site

how do we know which bacterial cells contain our gene of interest?

The lacZ gene encodes the β-galactosidase enzyme that will cleave the X-gal substrate into a blue product If a gene is inserted that disrupts the lacZ sequence, β-galactosidase activity is lost **the cells that are blue do not contain sequence of interest

Sanger method

The lack of a 3' hydroxyl group prevents addition of subsequent nucleotides during DNA synthesis uses chain terminator dNTPs These chain-terminating nucleotides lack a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. (stops further replication since there is no way to add more from that) ***ddATP terminates synthesis because the 3' H cannot act as nucleophile in phosphodiester bond formation We identify the chain terminating nucleotide by a specific fluorescent dye, 4 specific colors to be exact. Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end. ***an individual reaction is set up for each nucleotide ***the sequence does not include the primer when on the gel

Transcription factors and Pol II form the preinitiation complex at the promoter NEED TO KNOW

Transcription factors assemble sequentially at the core promoter sequence along with Pol II to create the preinitiation complex Binding of TBP (tata box binding protein) along with TFIID to the TATA box is one of the first steps; binding of TFIID initiates recruitment of other factors required to begin transcription ****TBP binds to and bends the MINOR groove of the TATA box DNA -stirrups bends minor groove and opens it up a little TFIID and TBP recruit TFIIB to the promoter TFIIB directs asymmetric assembly of the preinitiation complex **important because it directs direction (one direction or another) so have to have steps that are going to direct asymmetric assembly The ATPase activity of TFIIH melts the promoter and converts the complex to an open state

Transcription involves a transient interaction between what?

Transcription involves a transient interaction between substrate RNA and DNA template 1. Around 17 bp of DNA is unwound at any given time during transcription -Causes supercoiling**** -Pretty small bubble, so don't need to worry about needing a topoisomerase*** 2. Around 8 bp of RNA are base-paired with the template DNA at any given time during transcription 3. No exonuclease-based proofreading mechanism -RNA doesn't have an exonuclease, which is another reason it is way less accurate -Transiently pulled apart into single strands, one is a template strand (base pairs with growing rna molecule) and non-template strand (coding strand) Transcription proceeds from left to right As helix reforms, the rna strand is displaced and hanging out the back

Prokaryotic and eukaryotic transcription use a multi-subunit RNA polymerase... what are the parts?

Two alpha subunits, two beta subunits, one w subunit -looks like a claw The β and β' subunits form the "active center cleft" of the enzyme at the base of the "pincers" This is the active site of the enzyme where nucleotide addition occurs using a two metal ion mechanism (as with DNA polymerase) components of transcription pass through "channels" in RNA polymerase during elongation

Sequencing PCR reactions uses chain terminators in what amounts?

VERY SMALL amounts ***this is because everything would terminate at the smallest amount if there was an excess amount of ddATP For each segment of DNA being sequenced, we set up four sequencing PCR reaction - one with each of the four possible chain terminators Set up standard PCR reaction in PCR tube with template, primer, polymerase, dntp, mgcl and SMALL amount of all the ddATP Run pcr, get 5' to 3' extension, these reactions proceed normally but for every nucleotide incorporated in strand, there is a small chance of chain termination By time pcr is finished, have extremely heterogeneous product, terminated at different lengths Run these on gel to sort out; run on polyacrylamide gel (massive gel) Set up 4 reactions, each for each nucleotide; a has a chain terminators and so on, so there will be termination only at that specific nucleotide **this is question on exam

how would we find our favorite gene in a library?

We can use a modified Southern blotting and DNA-DNA hybridization protocol to identify our favorite sequence in a library Bacterial colonies are transferred to a membrane and the DNA is crosslinked DNA probes are used to detect our sequence of interest - then we can go back and find the colony that corresponds to that location

How to deal with degeneracy?

Wobble bases it helps protect from mutation -wobble ONLY happens in the 3rd position of codon, which means that it only causes mutations 25% of the time; more tolerant of mutations Changing first position still leads to similar AA (changes polar for another polar, etc)

Splicing occurs via what kind of reactions?

a pair of transesterification reactions First transesterification: 2' OH group on the branch point attacks 5' splice site Second transesterification: 3' OH group on 5' splice site attacks 3' splice site Intron is released as a lariat

What is polyadenylation directed by?

a transcribed signal sequence Coordinated by phosphorylation state of C-terminal tail of polymerase and by a transcribed signal sequence After transcribe all coding region of gene, transcribes target for polyadenylation factors: AAUAAA Polyadenylation factors: free transcript away from polymerase by endonuclease 5' end attracts xrn2 and RAT1 to start termination 3' end attracts poly-a polymerase: starts at 3' end of rna to add series of ATP nucleotides, that long string of a nucleotides recruits poly-a binding protein, which coats the poly-a tail Polyadenylation factors are recruited to the c-terminal tail of Pol II and then transferred to the AAUAAA sequence when it is transcribed The transcript is cleaved, and then polyadenylated by the Poly-A polymerase Poly-A tail is bound by PABP, which aids in the initiation of translation

What is special about the guanine base that is used for capping?

beta to alpha phosphate links the cap allows for a 5' to 5' linkage, which is the only time this ever occurs methyl groups added to specific C groups on guanicine base are used to tag as not a regular GTP -Because of this, you sometimes get 2' methylation on first couple nucleotides -Benefit: it decrease the chances that the reactive 2' OH groups lead to degradation (creates more stability)

chemically competent vs electrocompetent

chemically competent: bacterial cells are prepared using heat shock electrocompetent: prepare cells to be transformed with an electrical current

Illumina sequencing

done on a chip -Sequence, ligate adaptors to it, attach all of them to a chip that allows us to get tens of thousands of reactions to be happening at the same time -Flow in reagents one round at a time, -In a round of sequencing, flow in all four nucleotides, one get incorporated, observe fluorescence color depending on which got incorporated Drawbacks: very expensive (several hundred to thousand per reaction), requires template to start with that is VERY high quality, takes longer Positives: get a ton more data i. The DNA sample is fragmented, ligated to an adapter sequence, and adhered to a chip ii. We can then add fluorescently labeled dNTPs in a sequencing reaction to measure which nucleotides are incorporated into the islands and in which order - this will tell us the sequence of the DNA in each island iii. The dNTPs used are reversible chain terminators - chemical modification blocks the 3'-OH group, but this blocker can be washed off after each round of incorporation iv. This lets us make sure that only one nucleotide at a time is ever incorporated

chromatograms for sanger sequencing

each colored line on the chromatogram is called a trace, and represents the density of the four different nucleotide lanes at that point in the gel good way of sequencing 500-1000 bases of DNA; NOT to sequence genome -fast and inexpensive -drawback: don't know what primer to use; you have to start by knowing partial sequence to know the primer needed

RNA processing factors are recruited during ______.

elongation phosphorylation state changes during the process of transcription (recruit in different enzymes at different times to coordinate capping, splicing, and polyadenylation)

Where are mature mRNAs exported to?

exported to the cytoplasm through the nuclear pore complex. M mrna bound by exportin and ran protein; then is able to move through npc into cytoplasm Ran hydrolyzes gtp into gdp dissociates.. Look up Importin protein, ran binds in gdp bound form allows complex to go back inside ** if splicing hasn't happened, mRNA cannot be exported -protein tab left at each splice junctions (the exon junction complex) and they have to be there in order for export to occur

How do you read the gel for the Sanger sequencing reaction

from the bottom of the gel upward toward the well **gives sequence of growing strand, not the template if there was an excess amount of ddATP, everything would terminate at smallest amount

tRNA

fulfill the requirement of the adaptor hypothesis -short RNAs that fold up into very stereotyped hairpin structure -3' end covalently binds to amino acid -Bottom arm (anticodon arm) has loop with variable sequences that are the anticodon, can bind to different combos of base pairs in mRNA lets us add specific amino acids to growing peptide chain

degeneracy

have at least one tRNA for every amino acid, but do not have different tRNA for every possible codon aka the genetic code is this

Hoagland and Zamecnik demonstrated the ability of the tRNA to transfer amino acids onto a growing peptide strand using fractionation experiments

i. tRNAs linked to radioactive leucine amino acids were added to ribosomes and allowed to incubate ii. After spinning the reactions down, the tRNAs remained in the supernatant, while ribosomes and new proteins ended up in the pellet iii. Over time radioactivity was transferred to the pellet from the supernatant, indicating that the leucines were transferred from tRNAs to growing peptides

Why is TFIIB allowing asymmetric assembly of the preinitiation complex important?

it directs direction (one direction or another), so have to have steps that are going to direct asymmetric assembly

Which is denser? linear or loop

linear introns are denser than loop

Spliceosome

makes sure all reaction that happen during splicing happen accurately and efficiently at the same place every single time splicing is given specificity and is catalyzed by this consists of 5 small ribonuclear complexes called snRNPs: U1, U2, U4, U5, and U6 -each snRNP is a RNA molecule and a bunch of proteins

Rule of the genetic code

no gaps and no overlaps; read code straight through Shown a single nucleotide mutation only causes a change in one amino acid in protein; evidence of non-overlapping makes ORFs highly sensitive to frame-shift mutations- if one is changed, every codon that follows will change

how can topoisomerases be used to insert DNA sequences into plasmids?

plasmids can be purchased that have already been cut by Topoisomerase I, which still attached to the cut ends when Topoisomerase I re-ligates the plasmid, it wills ometimes incorporate our insert into the plasmid: TOPO cloning Mix activated with product, the vector and insert come together and then the topo then repairs one strand or other (ligate insert and vector together) -This can be transformed as normal -Doesn't depend on presence or absence of specific cut sites -More expensive

What is PCR?

polymerase chain reaction -used to synthesize DNA in a test tube (in vitro) rather than in the organism -helps to amplify a specific target region of DNA -leads to exponential amplification of DNA fragments (every round doubles the DNA amount in tube) **uses thermostable DNA polymerases to synthesize DNA

Possible undesired PCR products

primer dimers: if primers stick to each other too well, the primers themselves will become template for DNA replication; bc it is so small, it can replicate super easily, prevent desired product from replication amplification of sequence that comes from the incorrect primer binding

major way we might use molecular cloning for?

production of recombinant human insulin -Use human DNA, isolate insulin gene -At same time, extract plasmid DNA from E coli -Using cloning, insert insulin gene into plasmid to make recombinant, grow up those on massive scale... can then be purified and extracted

The adaptor hypothesis

proposed a link between mRNA and protein synthesis have mRNA of spec sequence, some kind of adaptor that interacts w mRNA on one side and amino acid on other to use that in order to decode mRNA into spec. series of amino acids Features expected to have: -complementary base pairs: gives us ability to bind RNA -a way to bind to the amino acid -a way to move down RNA tRNA filled this requirement!!

What kind of graph is made from qRT-PCR results?

results reveal an exponential increase in PCR product abundance

what is a problem with using restriction enzymes for cloning?

sequence cut sites pops up in the middle of sequence and cut gene in half limit what we can do because we don't want to cut the sequence of interest in half

Pyrosequencing

takes sample, break into pieces and ligate a DNA adaptor onto one end of DNA that a primer can adapt to -end up with short chunks of DNA with primer ligated, put these on plate -set up a bunch of sequencing reactions on plate, flow in reagents on plate to see what happens; flow in one nucleotide at a time Then, flow in dGTP, it is a watson-crick match, gets incorporated, and when it does, it releases pyrophosphate, which is converted to ATP by a sulfurylase enzyme, the ATP then interacts with luciferase, cleaves a molecule called luciferin that then emits light; idea came from fire flies Flash of light = incorporated nucleotide Sequences multiple pieces of DNA simultaneously problems: not always perfectly clean, constantly have to flow in each nucleotide separately so it takes a while i. The DNA sample is fragmented and ligated to an adapter sequence that provides a primer binding site ii. The fragments are loaded into wells on a plate and subject to PCR in the presence of luciferase and luciferin iii. As pyrophosphate is released by DNA polymerization, it will be converted to ATP and cause luciferase to cleave luciferin and emit light iv. By adding one type of nucleotide at a time (i.e. A,C,T,G) in stepwise fashion, we can measure which wells incorporate which nucleotide to determine the DNA sequence attached to each bead

Rho independent mechanism

the RNA forms a hairpin, and this secondary structure causes the transcript and template to pull apart ****doesn't require anything aside from transcript itself; uses features of the transcript to end transcription Requires 2 pieces: 1. sequence after stop codon that has dyad symmetry (inverted repeat); prone to forming a hairpin structure 2. Hairpin sequence puts strain on rna molecule; usually shortly after it is transcribe, polymerase will transcribe long series of uracil nucleotides Important to have bc uracil will bind to dna, they only have 2 hydrogen bonds, so it is easier to break bonds and pull molecule out

RNA polymerase holoenzyme

the assembled polymerase core and sigma subunit

How is the Sanger sequence semi-automated?

the dye labeled segments of DNA are applied to a capillary gel and subjected to electrophoresis Diff termination events should result in fragment that is labeled with dye color that corresponds to the dye that labeled the chain terminator; however, all are mixed together so have to run them through a capillary gel. Polyacrylamide gel through capillary tube; put all samples in there; they run through gel based on size/weight; for each fragment, the laser illuminates the fluorescent dye and the detector detects which light was emitted; computer does it for us, don't have to pour giant gel, only have to set up one reaction

What does log phase mean?

this is the phase you want cells to be in when you harvest them -mid log phase of growth means they are the absolute healthiest, plenty of nutrients are provided, able to divide rapidly they are the best able to be transformed

open reading frame

three potential ways to read frame depending on where you start -in most cases, only one will turn into a synthesis of a full-length protein bc the other two have random stop codons coding

how do snRNPs bind intron sequences?

through Watson-Crick base pairing (and some non-WC base pairing)

TFIIH

transcription factor arrives a little after Pol II, uses helicase activity to allow for melting to open the promoter

Group I self-splicing introns

use guanosine to execute the first transesterification step i. The intron RNA folds to produce a pocket that captures a "G" nucleotide ii. This captured "G" is brought in close proximity to the splice site by RNA folding iii. The 3' hydroxyl group of the "G" executes a nucleophilic attack on the 5' spice site for the first transesterification reaction iv. The second transesterification reaction occurs the same as in standard splicing **linear intron released

RT-PCR (Reverse transcription PCR)

used to amplify DNA from an mRNA template start with mRNA to use as template to make DNA copies of that mRNA -can synthesize DNA molecular from an mRNA template using a poly-dT primer that anneals to the poly-A region of mRNA Start with pool of mRNA molecules (they have long 3' poly-A tail) -Start out by adding a primer that has a bunch of T bases, form watson crick bp with poly a tail -Add reverse transcriptase to elongate and make copy

What is molecular cloning used for?

used to insert genetic material in a replicating system -helps us produce large amounts of specific DNA segments

SYBR Green qRT-PCR

uses a fluorescent dye that is incorporated into dsDNA uses a fluorescent dye that emits more fluorescence when it has bound to double-stranded DNA - thus the fluorescence emitted will depend on the amount of double-stranded DNA present

TaqMan qRT-PCR system

uses a fluorescent probed that is activated by polymerization which uses a sequence-specific probe that is labelled with a fluorophore and a quencher i. The quencher prevents the fluorophore from emitting light ii. PCR extension of the primer will remove the quencher from the probe and allow the fluorophore to emit light - thus fluorescence emission will serve as a measure of how much PCR extension happens at each cycle

How can recombinant plasmids be constructed?

using sticky ends left by restriction enzymes -compatible sticky ends can anneal to each other and form hydrogen bonds -DNA ligase enzyme can then be used to form phosphodiester bonds between the DNA molecules

What is chimera?

vector and DNA fragment together