Lecture 5: Elongation Flashcards
How does the initial polymerization occur? How is it inhibited?
Initial polymerization does not require movement of the enzyme.
• RNA synthesis begins immediately.
• The RNAP does not actually move as the first 9 nt are polymerised and it stays bound to the promoter.
• The transcribed DNA packs into the pocket of the enzyme, increasing the strain between the DNA and enzyme. Relaxation can be achieved by releasing the RNA (abortive transcription).
• Synthesis is inhibited by rifampicin
How does the RNAP move from initiation to elongation?
The release of σ is key to the transition.
• After synthesis of 9 nt enzymes stop making abortive transcripts.
• σ is released and the enzyme becomes resistant to rifampicin.
• Promoter contacts are broken.
• Displacement of the σ3.2 loop by the growing RNA transcript may cause promoter release through destabilising interactions between the β flap and σ4.
• This destabilisation would mean that σ4 and -35 interactions would be disrupted. RNAP can let go of the promoter and move along the DNA.
What are some traits of elongation?
Elongation is an extremely stable process which can occur for 1000s of nucleotides.
• TEC (tertiary/transcription elongating complex) contains the enzyme, DNA template and RNA.
• Error frequency is roughly 1 in 10,000.
• There is no obvious proof-reading (no 3’ to 5’ exonuclease).
What is the mechanism of elongation?
RNA synthesis occurs in a nucleotide addition cycle.
1) DNA and RNA are translocated so the 3’-OH of the RNA is in the I site of the RNAP.
2) NTP binds to the i+1 site.
3) A new phosphodiester bond forms.
4) Pyrophosphate is released.
What is the importance of the trigger loop helix? Draw a diagram
The trigger loop helix is important for the formation of the phosphodiester bond.
• When the bond formation step isn’t occurring, it is in a relaxed unfolded state.
• When NTP binds, the helix folds to form an α-helical hairpin (folded trigger helices).
• The helix has arginine and histidine residues required for correct orientation of the NTPs through hydrogen bonds.
• This cycle is required for rapid nucleotide addition.
• The TL/TH contributes to accuracy by easing the barriers to backtracking.
• Mutational analyses indicate that the conformational change can be rate-limiting and this reflects the ability of the incoming NTP to bind to TEC.
Why is pausing important during elongation?
The transient pause gives the RNAP 3 options.
1) Continue elongation.
2) Backtrack. This can be in response to DNA damage or mis-incorporated nucleotides.
3) Terminate the transcript by dissociating.
These pauses can sometimes be longer as well.
• Sometimes unwinding of downstream DNA may require more energy than usual while rewinding of upstream gives less energy than usual. This may mean that elongation speed is reduced from 60nt per second to less than 1 nt per second.
• A second reason for a longer pause is that base-pairing is weaker in the DNA hybrid than upstream. dA:rU is weaker by 200 fold than the other possibilities. In this case the transcription bubble returns upstream while the downstream rewinds. The enzyme follows the bubble upstream, leading to backtracking.
How does RNA polymerase backtracking occur?
RNA polymerase backtracking is occasionally required to remove damaged or misincorporated nucleotides.
• The RNA is repositioned and the 3’ end of the RNA is removed from the active site.
• The 3’ end of the RNA is cleaved to give a new 3’-OH.
• Backtracking occurs on average once every 100 bases.
• This is part of the reason why the error rate is so low (1 in 100,000).
• Short backtracks can activate the nucleolytic activity of RNAP to remove a dinucleotide.
• Further backtracks are prevented by a gating tyrosine.
• Recovery from a backtrack pause requires conserved transcription elongation factors.
How do elongation factors work?
Elongation factors stimulate overall transcription rates. They have a number of functions.
• Increase fidelity.
• Increase rate of elongation.
• Rescue arrested complexes.
• Stimulate promoter clearance.
• Modulate RNAP behaviour at pause sites.
• GreA and GreB are both positive elongation factors.
The elongation factors enhance the native cleavage activity of the RNAP.
How do longer backtracks occur?
When Pol II encounters a weak hybrid, it needs to backtrack further than the gating tyrosine.
• The increase in energy balance can help to overcome the gating tyrosine.
• Extensive backtracking traps RNA and the trigger loop in the pore, inhibiting elongation and arresting pol II.
• GreB arrests pol II by locking the trigger loop away from RNA and displacing the backtracked RNA.
• It complements the active site with a basic and two acidic side chains. This induces cleavage and release of backtracked RNA and creates a new RNA 3’ end.
