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27 Cards in this Set
- Front
- Back
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What is the transcription unit? |
transcription unit = DNA sequenceextending frompromoter toterminator & istranscribed into asingle RNA |
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E.coli RNA polymerase structure? what does it require? |
The holoenzyme is comprised of:α2ββ ́σ • enzyme assembly and promoterrecognition: two α subunits • the catalytic center is β+β ́ • σ factor is responsible for promoter recognition • The RNAP holoenzyme requires: - dsDNA template- NTPs and Mg2+ |
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sigma factor does what to loose/tight binding sites? |
decreases core K association at loose, promotes tight core binding at proper sites |
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How does RNA polymerase/promoter binding work? |
holoenzyme searches for promoter, finds it and binds loosely = closed promoter complex, sigma factor promotes tight binding of RNAP to promoter = open promoter complex, |
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Describe Methylation Interference (reverse footprinting), whats its main purpose? |
1) ) mild DMS treatment (2) methylation blocks protein binding (e.g. RNA pol) to DNA (3) unbound DNA passes through filter (4) methylated DNA in filtrate show bases important for binding (how? Depurination at methylated sites results inbreak in DNA) main purpose is to see what basepairs are important in binding |
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Whats the difference between DOWN and UP mutations? |
Mutations that destroy matches with the consensus sequence (make thepromoter weaker, resulting in less transcription) are called DOWN MUTATIONS, divergences from -10 or -35 consensus seq or insertions that increase distance between -10and -35 elements beyond 17bp Mutations that make the promoter sequences more like the consensussequences usually make the promoter stronger, are called UP MUTATIONS, changes that better match the -10 or -35 consensus seq or deletions that bring the distance between -10 and -35 closer to 17bp (increased transcription) |
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what are the Conserved regions of E. coli promoters? |
-10bp from start = TATABOX -35 bp from start = TTGACA consensus UP elements: in active promoters; part of extended promoters • PROMOTER MUTATIONS DEFINE IMPORTANT SEQUENCERESIDUES: up-regulating and down-regulating mutations |
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What is DNase footprinting used for? what kind of DNA does it use? |
used to investigate DNA/protein binding, end-labeled DNA (1 strand only) |
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Does the a subunit in RNA polymerase contact the UP element? |
In strong promoters it does, only parts of RNAP not in used by regular core promoter binding though |
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What is the CTD? |
its the RNAP alpha subunit that has an independently folded C-terminal domain (CTD) that can recognize and bind to a promoter's UP element, it allows for a very tight binding betwen RNAP and promoter |
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what are the four conserved regions of the signma factor? |
Region 1: prevents whole sigma polypeptide from binding DNA Region 2: critical for sigma activity; subdivided into 4 parts, region 2.4 recognizes tata box region 3: Core and DNA binding Region 4: subdivided into two parts; plays a key role in -35 box recognition |
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what region is homologous amongst E.coli and B.subtilis? |
Region 1, to prevent sigma from binding itself to DNA |
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How can promoter mutations be suppressed? What does this show about their relationship? |
by compensating sigma subunit mutations, Such mutations indicate contact points between σ protein andpromoter sequence. |
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What happens once RNAP binds to the promoter in closed complex? what is some experimental evidence of this? |
it melts 10+bp of promoter DNA forming open complex "transcription bubble", 1. elevated temperature stabilizes melted DNA and alsostabilizes RNAP- promoter interaction |
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Describe DMS footprinting |
Starts the same way as DNasefootprinting but then methylatewith DMS • DMS methylates (adds CH3groups) to adenines in DNA • The protein is then dislodged andtreated with piperidine, whichremoves methylated purinesresulting in apurinic sites, thenbreaks the DNA at these apurinicsites • The DNA fragments are thenelectrophoresed and the gel isautoradiographed to detect thelabeled DNA bands 29 Each band observed in the gel endsnext to a nucleotide that wasmethylated and thus unprotected bythe protein |
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What does DMS footprinting show about protein binding? |
binding the proteindistorts the DNA double helix such thatit makes the base corresponding to thisband more vulnerable to methylation,i.e. melting (DMS methylates ssDNAmore willingly) |
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How do we know that early promoter binding by RNAP melts DNA? Explain |
Methylation-S1 nucleaseassay: that shows that these sizes tell how far the melted DNA region was from the labeled DNA end. |
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Describe METHYLATION-S1 NUCLEASE ASSAY, example? |
• promoter DNA end-labeled with 32P • dA exposed on ssDNA byRNAP promoter melting:susceptible to methylationby DMS • dA-Me cannot base pairwith dT: stays single-stranded and S1nuclease (cleaves onlyssDNA) sensitive • fragments resolved on gelat open, “melted” regionsand sequenced • These sizes tell how far the melted DNA region was from the labeled DNA end. example: RNAP melts phage T7 A3 promoter DNA inthe -9 to +3 region: |
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What are the stages to transcriptional recognition and initiation? |
1. RNAP binds to the DNA in a closed promoter complex 2. sigma factor stimulates polymerase to convert closed promoter to open promoter complex 3.polymerase incorporates the first 9 or 10 nt into nascent RNA (some abortive transcripts are made) 4. polymerase clears the promoter and beings the elongation phase, the sigma factor comes off the core RNAP |
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whats the basic summary of the experiment that demonstrated abortive transcripts? |
polymerase treated with heparin to lock RNA polymerase onthe DNA to see that it kept producing short abortive transcripts Many oligos per polymerase, thus many abortive transcripts madewhile RNAP bound to the promoter! |
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Describe the FRET analysis experiment and what they discovered/ruled out |
FRET analysis to discover mechanism of abortivetranscription,By changing where the donor and acceptor was placed, transientexcursion and inchworming models were ruled out, while the scrunching model wasconfirmed3 different models: trailing edge (if this washappening, they would have saw the twoparts of the fret signals, they didn’t see a difference in signal), leadingedge (inchworming: if this was happening, the two floors would be sepereatedand you would see a signal), downstream and promoter DNA(scrunching)(you canprove: by putting donor and acceptor on the DNA, on promoter and downstream oftranscriptional start: u will first see signal to start off with, as its pulledin, it is quenched, this is exactly what they saw). |
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how are abortive transcripts made? |
DNA scrunching |
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Describe Elongation phase in terms of what RNAP does and what subunits are necessary |
it synthesizes RNA 5' to 3' moving the transcription bubble along the template, beta and beta prime are required for addition of new NTPS to the growing RNA transcript |
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how does the phosphodiester bond form? |
Similar to DNA, RNA synthesized byaddition of NTPs to the 3’OH, Growing RNA chain forms shortRNA-DNA hybrid in unwound regionbut RNA emerges as single freestrand |
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Topology of dsDNA transcription |
combination of RNAP rotates itself around transcribed DNA, no super coils but tangles itself with DNA duplex and Template DNA rotates -unwinds ahead of RNAP,winds behind RNAP |
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what are the two types of transcription terminators in E.coli? Describe them |
1. intrinsic terminators (rho independant): RNA pol stalls at terminators (string of U-A pairs), causes transcription to pause and hairpins to form, these pull RNA out of the polymerase and transcription bubble to collapse,2. extrinsic terminators (rho dependant) requires RHO factor to terminate: Rho binds to RNAP in the elongation phase, as it grows RNA enters Rho's hexameric ring, it continues to feed through the ring until a terminator sequence where it will then pause and trap the elongation complex, Rho (ATPase) then unwinds the RNA-DNAhybrid within the transcriptional bubble,releasing the RNA and terminatingtranscription |
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describe Rho structure and abilitiy |
hexameric ring of identical subunits, hydrolyzes ATP, has RNA-DNA helicase activity |
