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In which organisms is transcription coupled with translation?
Prokaryotes
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what is the difference between eukaryotes and prokaryotes?
- Nucleus!
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Define Transcription
- the synthesis of RNA, using DNA as a template
- occurs in a 5' to 3' direction
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What can RNA Pol do, that DNA Pol cannot?
- RNA Pol can:
- make RNA de novo (i.e. it does not need a primer)
- has limited proofreading capacity
- incorporates NTPs instead of dNTPs
- U instead of T
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Which direction does Transcription occur in?
- Read 3' to 5'
- Written 5' to 3' direction
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Which DNA strand is a gene transcribed from?
template strand
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Which DNA strand is a gene translated from?
- coding strand
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What is meant when transcription is described as asymmetric?
- The transcription direction on different strands is opposite
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Where is the Pribnow box located?
-10 element (consensus sequence TATAAT)
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What are the two discrete DNA elements prokaryotic promoters consist of?
- -10 Pribnow box (TATAAT)
- -35 tcTTGACAt
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for prokayrotic transcription, which is more important: the absolute sequence of the spacer region between the -35 and -10 boxes or the length between?
- the length between is more important (11-18 bp)
- spacers that are longer or shorter make weak promoters
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Compare the frequency of trancription for genes with strong vs. weak promoters:
- strong promoters: as often as every 2 seconds
- weak promoters: about once every 10 minutes
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Fill in the blanks:
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What are the activities of prokaryotic RNA Polymerase?
- Scan DNA and identify promoters
- Initiate transcription
- Elongate the RNA chain
- Proofread
- Terminate transcription
- Be responsive to regulatory proteins (activators and repressors)
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What are the RNA holoenzyme subunits?
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What experiment did Hinkle and Chamberlin perform?
- looked for affinity of RNA pol for DNA in vitro
- measured the rate of dissociation of RNA polymerase-DNA complex
- filtered so that only labeled DNA stayed on the filter if it was bound to RNA polymerase
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Compare the binding ability of Holoenzyme to promoters vs. Core RNA Pol to promoters
- The holoenzyme binds to promoters tightly, while core elements disaccociate
- sigma factor stimulates tight binding between RNA polymerase and promoters
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What ensures that the RNA Pol holoenzyme binds to promoters?
sigma factor
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How does the sigma factor change the DNA binding properties of RNA pol?
σ factor changes the DNA-binding properties of RNA pol so that its affinity for general DNA is reduced and its affinity for promoters is increased
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At what point does the σ factor fall off?
- The σ is only required for initation of transcription and does not participate in elongation
- released when the nacent RNA chain reaches ~10bp
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Which molecular technique has enabled localization and identification of the sites to which RNA pol binds at promoters?
DNase I foot-printing
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What do the β elements of RNA Pol act as?
β clamps/crab claw
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Where is the active site (site that is being transcribed) located within RNA polymerase?
The base of the two "pinchers" forming the beta clamp
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RNA Polymerase Holoenzyme:
β and β' subunits
- make up the most mass of the enzyme and form the catalytic center containing-
- the main channel through which DNA passes during transcription
- the secondary channel through which NTPs enter the enzyme on the way to the active site
- the exit channel through which nascent RNA leaves the enzyme
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RNA Polymerase Holoenzyme:
α subunit
- There are two alpha subunits
- Serve as a scaffold for the enzyme core
- bind the promoter
- interact with the activator/repressor proteins
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RNA Polymerase Holoenzyme:
ω subunit
- plays a regulatory role
- participates in enzyme assembly
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RNA Polymerase Holoenzyme:
σ factor
required for promoter recognition
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What is the general rule relating the sigma factor to the lifestyle of bacteria?
- Usually, the more diverse the lifestyle of bacteria is, the more s factors the bacteria have
- E.coli has seven σ factors
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What does the α subunit interact with?
- the UP element
- a-CTD binds to the UP element, contributing to the very tight binding between RNA pol and promoter -> a high level of transcription
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The three stages of transcription:
- initiation
- elongation
- termination
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What does the antibiotic Rifamycin influence in RNA transcription?
- The initiation stage
- Rifamycin binds to the β subunit of RNA pol and blocks binding of the first NTP
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Transcription initiation in E.coli: Finding and binding the promoter
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Transcription initiation in E.coli: Closed Complex formation
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Transcription initiation in E.coli: Open complex formation
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Transcription initiation in E.coli: Binding of 1st NTP
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Transcription initiation in E.coli: Addition of the next NTPs
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Transcription initiation in E.coli:
Dissociation of sigma
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What is within the region known as the "transcription bubble"?
- The region containing RNA polymerase, DNA, and nascent RNA is called a transcription bubble because it contains a locally melted “bubble” of DNA
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Which stage of transcription is susceptible to cordycepin?
Transcription elongation
Cordycepin is a nucleoside that can be phosphorylated in vivo to give 3'-deoxyadenosine 5'-triphosphate
RNA pol can add this modified nucleotide to the growing RNA
However, because cordycepin lacks a 3'-OH, it aborts further elongation
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What are two auxiliary proteins that are utilized if transcription elongation is halted because of a missing or incorrect NTP?
- GreA, GreB
- these stimulate inherent RNase activity to cleave off a few nucleotides
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Describe the two methods of transcription termination in E.coli
- r (rho)-dependent terminators require assistance of an auxiliary factor Rho
- intrinsic (rho-independent) terminators abort transcription by themselves (without the help of any additional factors)
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How does Rho-independent transcription termination occur?
- Inverted repeats have a tendency to form a stem-loop (hairpin) structure
- Thought to destabilize the RNA-DNA hybrid transcriptional bubble
- A-U rich terminal sequence is less stable
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How does Rho-dependent termination occur?
Rho is an ATP-dependent hexameric helicase
It loads the RNA at the C-rich sequence (called Rho-utilization (rut) site) and migrates in 5’-3’ direction “chasing” RNA Pol in an ATP-dependent manner
When RNA Pol stalls upon encountering a termination site, Rho catches up with RNA Pol, unwinds the RNA-DNA hybrid, and releases the RNA chain
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