Term
| What is needed for transcription? |
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Definition
1.RNA polymerase
2.DNA template
3.Ribonucleotides (rNTPs)
4.Regulatory sequences (promoters and terminators to control gene expression) |
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Term
| What is the Travers and Burgess experiment in 1969? |
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Definition
| They did a SDS-PAGE of RNA polymerase from E. coli and it showed several subunits. |
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Term
| What are the subunits that Travers and Burgess discovered of RNA polymerase? |
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Definition
Beta (150kD) and Beta'(160kD)
Sigma 70kD
2 Alpha 40kD
Omega 10kD (not seen in this experiment but later discovered) |
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Term
| What role does omega play in? |
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Definition
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Term
| Can the cell stay alive without omega? |
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Definition
| Yes, it is not required for cell viability or in vivo enzyme activity |
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Term
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Definition
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Term
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Definition
| The holoenzyme without Sigma |
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Term
| What happens when the core and sigma are separated? |
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Definition
| the core will transcribe promiscuously |
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Term
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Definition
| Binding sites for RNA polymerase where specific transcription is initiated |
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Term
| What is the big theme in this class? |
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Definition
| interaction b/w specific protein and specific dna sequence and how they interact with other proteins (recruit other proteins to regulate proceses) |
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Term
| Fig 6.3. What is the experiment that Hinkle and Chamberlin did and what did they conclude? |
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Definition
| The setup: Filter binding Assay. Add labeled T7 phage DNA to compete with unlabeled DNA. Do this test with and without sigma to show that sigma does promote tight binding at least in certain DNA sites |
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Term
| What were the conclusions brought on by the Hinkle and chamberlin experiments (Holoenzyme Binding)? |
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Definition
1. Sigma factor promotes tight binding at certain DNA sites
2. Separate experiments shows that the holoenzyme (like the core) has weak binding sites on the DNA
Therefore: HOloenzyme can interact with template DNA at both tight and loose binding sites whereas the core only interacts only loosely |
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Term
| What are the tight binding sites? Loose? |
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Definition
Tight: Promoters
Loose: the rest of the DNA |
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Term
| What can initiate transcritpion immediately? |
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Definition
| Tight complexes b/w the Holoenzyme and T7 DNA |
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Term
| How many tight binding sites compared to loose are there on the promoters? |
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Definition
| Only 8 tight binding sites and 1300 Loose sites |
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Term
| When is tight binding enhanced? Why |
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Definition
at Higher temps
Because the DNA is denatured (unwound) so the promoters work better at transcritption |
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Term
| Why does the Tight site = promoter theory make sense? |
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Definition
| If the tight sites are promoters, then the inability of the core to bind to the tight sites explains its inability to transcribe DNA specifically |
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Term
| When does the Holoenzyme bind more tightly to the T7 DNA? Think figure 6.4 |
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Definition
| when the temps are elevated (namely holds on better at 37*C compared to 15*C) |
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Term
| What are common sequences to ALL ecoli promoters? |
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Definition
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Term
| What sequence does the core promoter usually have or start with what letter? |
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Definition
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Term
| What are the Core elements of the promoter? |
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Definition
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Term
| What are some additional regulatory elements of the promoter? |
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Definition
| Up element--RIch in A- and is further upstream (-40 to -60) |
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Term
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Definition
| Enhancers (not classical promoter element b/c they dont bind RNA pol directly) |
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Term
| Where are Fis sites located? |
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Definition
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Term
| What are teh binding sites for the Fis protein? |
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Definition
| Transcriptional activator |
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Term
| Look at figure 20.4 what happens if one of the components is missing? |
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Definition
| something will go wrong in transcription |
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Term
| What did Carpousis and Gralla find in their work with Transcrition INitiation? |
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Definition
| Very small oligonucleotides (2-6 NT long) are made without RNA polymerase leaving the DNA. These are abortive transcripts and can be up to 10 Nt |
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Term
| In the Transcription INitiation, what else did Carp and Gralla add to determine what happens in their experiment besides the typical things needed for Trasncritption? Why? |
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Definition
| Heparin--b/c it binds to any free or unbound RNA polymerase. As soon as RNA polymerase leaves the DNA it is bound to Heparin taking it out of the "game" |
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Term
| What are the stages of transcription initiation? |
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Definition
1. Forming the closed promoter complex (polymerase binds loosely)
2. Forming the open promoter complex (sigma will tightly bind here) starting transcription
3. INcorporating the first few nucleotides
4. Promoter clearance (the transcript is long enough to be extruded from the polymerase and sigma is released b/c no longer needed |
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Term
| What are the little green things in figure 6.9? |
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Definition
| Transcripts that were too short to remain stably bound to template, the polymerase will start over until the RNA "catches" |
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Term
| What are the steps for transcription initiation? |
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Definition
1.formation of closed complex at promoter
2.formation of open complex (unwinding/melting)
3. polymerization of first 9-10 nucleotides are polymerized while polymerase remains at promoter
4. Promoter clearance:
a. Transcript forms stable hybrid with template
b. Polymerase adopts "elongation" conformation and moves away from the promoter (sigma is release) |
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Term
| What does sigma stimulate? |
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Definition
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Term
| How does sigma stimulate transcription initiation? |
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Definition
1. Sigma directs tight binding of RNA polymerase to promoters
2. Sigma directly stimulates transcrition initiation
3. Indirectly, sigma enhances elongation by stimulating initiation. |
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Term
| Describe an experiment that proves that sigma stimulates transcription initiation. |
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Definition
| Again, Travers and Burgess conduct an experiment. They added sigma at different concentrations and measured the rate of transcription with radioactive nucleotides |
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Term
| What were the results of Travers and Burgess's transcription initiation experiment? |
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Definition
| The more sigma, the more initiation and syntehsis we see. |
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Term
| Is sigma recycled or is a new sigma used in each transcritption event? |
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Definition
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Term
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Definition
During initiation sigma can be recycled for additional use in a process called the sigma cycle.
Core enzyme can release sigma which then associates with another core enzyme |
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Term
| Describe an experiment that proves that sigma can be recycled fig 6.11 |
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Definition
Start Experiment at time 0 (xaxis) and it begins and they have template DNA and polymerase (ecoli RNA comes from strain that is sensitive to an antibiotic called Rif^s or Rifampicin) the antibiotic blocks transcription initiation.
This experiment is done under low salt conditions so it discourages disociation of RNA polymerase from DNA.
At Minute 10 Add core that is either +Rif or -Rif proving that sigma core that is not resistant to Rif can be attached to a +Rif Core Green=enzyme under stress, new core (resistant) and old sigma (not resistant) |
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Term
| What has the resistance to Rif? |
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Definition
| The core has the resistance to Rif so when an old sigma can attach to a resitant core in +Rif transcription still occurs |
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Term
| What are 2 pieces of information we can gain from the Recycling Sigma experiment? |
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Definition
1. Sigma is recycled because it is released
2. Core has antibiotic resistance. |
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Term
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Definition
| Upon promoter clearance, sigma dissocaites from RNA polymerase and joins new core to initiate another RNA chain. |
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Term
| Does Sigma actually get recycled? |
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Definition
| Maybe not... there is another model |
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Term
| What did Roberts et al. show in 1996? |
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Definition
| that sigma is still attached to core at positions +16/+17 well after promoter clearance has occured |
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Term
| Why did T&B's experiment work? |
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Definition
| Their model involved harsh separation techniques that could strip sigma off the core if it is weakly bound |
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Term
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Definition
| Flurescnence Resonance Energy Transfer |
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Term
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Definition
| You need 2 probes: a Donor (on sigma) and an Acceptor (on DNA)and you measure the proximity b/w the 2 probes |
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Term
| What is important about the acceptor Probe? |
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Definition
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Term
| If Efficiency goes ____ probes are close, if efficiency goes ____ probes are too far |
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Definition
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Term
| Does FRET involve harsh separation techniques? |
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Definition
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Term
| What kind of FRET is most useful for our purposes? Which one does not distinguish b/w dissociation or no dissociation of sigma? |
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Definition
| Leading Edge fret is useful. Trailing edge FRET does not distinguish b/w dissociation or no dissociation of sigma |
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Term
| Why is Trailing edge fret useless for our purpose? |
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Definition
| Whether sigma is released or not released doesnt matter because the distance b/w the donor and acceptor will increase regardless (6.13a) Increase distance=decreased FRET. Either way, FRET is decreased and this is useless |
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Term
| Why is Leading edge fret useful? |
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Definition
| Decrease in FRET efficiency if sigma is released. If sigma is not released, FRET will be increased in energy(probes get closer. If sigma is released Fret will be decreased (distance increases) FRET goes down. |
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Term
| What does trailing edge fret tell us? 6.14 |
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Definition
| Nothing, the energies dropped either way in both cases |
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Term
| What does leading edge FRET tell us? |
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Definition
Results: energy increases
CONCLUSION: so the distance between the two probes decreasing showing that sigma is not released (remains associated) |
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Term
| So what is the conclusion and Current model for Sigma? |
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Definition
1.sigma does not dissociate from the core during elongation
2. Sigma changes its relationship to/affinity for the core (sigma may be dissociated if it changes its affinity for core)
3. Sigma may be recruited by another core |
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Term
| Describe a study that determines where the actual melting of the Base Pairs occurs during transcription. |
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Definition
Siebenlist et al.
Melt the Base pairs, Add DMS and see where the DMS formed and blocked the reformation of the H bonds. |
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Term
| Describe the DMS melting experiment thoroughly. 7 steps |
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Definition
1. Start with T7 labeled promoter.
2. Bind RNA polymerase
3. Melt a particular region forming an open complex by adding a polymerase.
4. Add DMS (this will methylate all the free A's )
5. Remove polymerase
6. Use S1 to cut single stranded DNA
7. Run a gel to see where the S1 nuclease cuts the single stranded DNA |
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Term
| What happened to the results in Siebenlist's experiment? |
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Definition
| There was a big smear that indicated more than just the single adenines were unable to form Base-Pairs, the neighboring nucleotides also remained single stranded |
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Term
| How big is the melted region? |
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Definition
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Term
| What were the four lanes of in the PAGE Gel? (describe the gel) |
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Definition
FIRST LANE: R+S- (first lane) NOT including S1 Nuclease so it is not chopped up at all and it ended in step 4
SECOND LANE: R+S+ (including S1 Nuclease) full expermeint [what we are interested in] you can see the size of the bubble here
Goes from -9-->+3. Size is about 12 nucleotides
This is about 12-17 nucleotides
THIRD LANE : NO polymerase ( you have the first initial piece of DNA because nothing was cut [step 1])
FOURTH LANE: R-S- (nothing was cut or no methyl was added) |
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Term
| How big is the "moving" transcription bubble where active initiation/elongation occurs? |
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Definition
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Term
| In 1970 what did Heil and Zillig find out about polymerase subunits? |
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Definition
| They separated and purified subunits |
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Term
| What did Heil and Zillig want to find out? |
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Definition
| which subunit has the resistance to Rifampicin |
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Term
| Which subunit has the Rif resistance? How was this determined? |
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Definition
| Beta subunit, determined by mixing and matching different subunits refer to figure 6.28 |
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Term
| What are the other roles that the beta subunit plays? |
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Definition
Involved in Rif Resistance and Therefore must have role in initiation of transcription
Also involved in streptolydigin resistance/sensitivity which blocks elongation of transcription and does not effect initiation |
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Term
| What do initiation and elongation have in common? |
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Definition
• During initiation we have polymerase binding to promoter (this does not happen in elongation)
• Melting in strands (maybe also in elongation)
• Chain elongation occurs in initiation and elongation |
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Term
| What did Grachev et al seek to discover? How did they accomplish this ? |
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Definition
| show evidence that the beta subnuit is involed in Phosphodiester bond formation. This was done by using affinity labeling |
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Term
| Describe Affinity labeling. |
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Definition
| Use a labeled substrate (ATP) analog that will form a covalent bond to the enzyme active site, then you can locate the label on the active site on the enzyme |
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Term
| What is a known problem with affinity labeling? |
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Definition
| Reagent 1 will form a covalent bond with amino groups in RNA polymerase's active site, but it might attach to other amino groups on the protein, not just the active site so the entire enzyme will be labeled |
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Term
| What is a workaround to the problem with affinity labeling? |
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Definition
| Use a radioactively labeled nucleotide (32P-UTP) that will form a phosphodiester bond bond with the affinity reagent/analog in the active site only. then you dissociate the subunits and look for the label |
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Term
| Where did they find the radioactive label? |
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Definition
| n beta subunit (implying where active site is) b/c that’s where the label is retained |
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