How fast should a polymerase's checkpoint steps be?
In models of an RNA-copying enzyme, the steps that tell right from wrong nucleotides must run at a moderate speed, since both too fast and too slow raise the error rate.
Source
Balancing Non-Equilibrium Driving with Nucleotide Selectivity at Kinetic Checkpoints in Polymerase Fidelity Control
Study at a glance
- Design
- Computational / modelling — Steady-state solutions of three-state and five-state chemical master-equation models of the nucleotide addition cycle, varying one rate at a time.
- N
- No sample; results are model calculations using T7 RNA polymerase rate parameters from earlier experiments.
- Population
- Kinetic models of T7 RNA polymerase transcription elongation
- Outcome
- Elongation rate, error rate, entropy production and heat dissipation per cycle
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What they did
The authors built three-state and five-state kinetic models of the cycle by which an RNA polymerase adds one nucleotide, splitting the paths for correct and incorrect nucleotides. Using rate constants taken from earlier measurements on T7 RNA polymerase, they solved for the non-equilibrium steady state while changing one transition rate at a time. For each setting they computed elongation speed, error rate, entropy production and the free energy spent per cycle.
What they found
Speeding up steps that do not discriminate between nucleotides (binding, translocation) pushed the system far from equilibrium and drove the error rate down to a floor near 6 × 10^-4. Steps that do discriminate behaved differently: very fast addition raised errors toward 10^-2, while very slow addition moved the system near equilibrium where errors also rose. The measured T7 rates for nucleotide addition and unbinding sit close to the error-minimising value. In the five-state model, selection dominated by the pre-insertion state fitted the T7 parameters better than post-insertion selection.
The limits
What it doesn't show
This is a purely theoretical model with no new measurements, and several parameters (such as the catalytic rate) were estimated rather than measured. Selection strengths were held fixed at chosen values, so the results show trends rather than predictions for real error rates. The model ignores proofreading, sequence dependence and frameshift errors, and the authors note that which step is truly the checkpoint in T7 RNA polymerase is still unknown.
Key terms
- Non-equilibrium steady state (NESS)
- A state where probabilities stay constant but a net flux flows through the cycle because energy is continually supplied.
- Kinetic checkpoint
- A transition in the cycle where correct and incorrect substrates have different rates, allowing wrong ones to be rejected or slowed.
- Chemical master equation
- A set of equations giving how the probability of each state changes over time from the transition rates between states.
- Entropy production
- A measure of irreversibility per cycle, zero at equilibrium and positive when the process is driven.
- Error rate
- The fraction of the net flux that incorporates wrong nucleotides.
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Quiz yourself
What happens when a non-discriminating step such as translocation is sped up?
Common questions
Why doesn't making every step faster reduce errors?
For steps that discriminate, going faster leaves less time to reject wrong nucleotides, so accuracy falls even though the system is strongly driven.
Why are errors high near equilibrium?
With little free-energy drive, the small energy difference between right and wrong nucleotides cannot suppress mistakes, and an entropy-driven regime favouring wrong additions appears.
Is this based on experiments?
It uses rate constants measured in earlier biochemical and single-molecule studies, but the paper itself only reports model calculations.
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