Balancing Non-Equilibrium Driving with Nucleotide Selectivity at Kinetic Checkpoints in Polymerase Fidelity Control

Abstract

High fidelity gene transcription and replication require kinetic discrimination of nucleotide substrate species by RNA and DNA polymerases under chemical non-equilibrium conditions. It is known that sufficiently large free energy driving force is needed for each polymerization or elongation cycle to maintain far-from-equilibrium to achieve low error rates. Considering that each cycle consists of multiple kinetic steps with different transition rates, one expects that the kinetic modulations by polymerases are not evenly conducted at each step. We show that accelerations at different kinetic steps impact quite differently to the overall elongation characteristics. In particular, for forward transitions that discriminate cognate and non-cognate nucleotide species to serve as kinetic selection checkpoints, the transition cannot be accelerated too quickly nor retained too slowly to obtain low error rates, as balancing is needed between the nucleotide selectivity and the non-equilibrium driving. Such a balance is not the same as the speed-accuracy tradeoff in which high accuracy is always obtained at sacrifice of speed. For illustration purposes, we used three-state and five-state models of nucleotide addition in the polymerase elongation and show how the non-equilibrium steady state characteristics change upon variations on stepwise forward or backward kinetics. Notably, by using the multi-step elongation schemes and parameters from T7 RNA polymerase transcription elongation, we demonstrate that individual transitions serving as selection checkpoints need to proceed at moderate rates in order to sustain the necessary non-equilibrium drives as well as to allow nucleotide selections for an optimal error control. We also illustrate why rate-limiting conformational transitions of the enzyme likely play a significant role in the error reduction.

Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control

“…On the other hand, biochemical and kinetic studies have demonstrated that a slow step follows the initial NTP binding to allow for the nucleotide insertion (25). We have also shown generically that the slow or rate-limiting step can play a significant role in the fidelity control of the template-based polymerization (26). It is highly likely that the non-proofreading T7 RNAP relies on the slow nucleotide insertion to conduct substantial nucleotide selection, or transcription fidelity control.…”

“…Following the previous clues (22), both an on-path and an off-path insertion processes of the non-cognate nucleotides were probed for the free energy calculations. Finally, we were able to infer the selection free energetics down to the catalytic stage by fitting with experimentally measured error rates via a chemical master equation (CME) approach onto the T7 RNAP elongation kinetics (26,31). This way, we completely characterized the fidelity control in this prototypical viral transcription system, with both classical structural dynamics and free energetic details.…”

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

“…We have recently studied transcription elongation of T7 RNAP by combining physical modeling and all-atom MD simulations, addressing both mechano-chemical coupling and fidelity control mechanisms during elongation [ [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] ]. The mechanochemistry concerns about how the protein machine utilizes chemical free energy to generate mechanical or directional motions, referring to how the chemical synthesis of RNA couples with the polymerase translocation along DNA in the RNAP system.…”

“…The nucleotide insertion accompanied by a conformational transition from a ‘semi-open’ to the closed form has been suggested to be a slow step during the NAC in T7 RNAP from previous biochemical studies [ 22 ]. It is highly likely that the slow insertion step is employed by the enzyme to substantially scrutinize against non-cognate nucleotide species to achieve sufficiently high transcription fidelity [ 35 ]. In viral T7 RNAP elongation, an elongation error rate reaches to ~10 −4 to 10 −6 for the base mismatch incorporation (e.g.…”

“…Upon the pre-insertion rejection and further insertion inhibition, the elongation error rate (i.e., for rGTP replacing rATP) would be reduced to ~ 10 −3 , according to our calculations via a chemical master equation (CME) approach [ 35 , 68 ]. Hence, to reduce the error rate further down to ~ 10 −4 , selection into the catalytic stage seems to be required, for which we estimate that a selection energy ~ 7 k B T is necessary.…”