A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

Abstract

RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for "selective ratcheting". The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.

Keywords: Fidelity control; Nucleotide selection; PPi release; RNA polymerase; Translocation.

Fig. 1

The PPi release and translocation mechanism of T7 RNAP revealed from extensive MD simulations and the MSM construction [31,34]. (A) Left panel: A molecular image of T7 RNAP elongation product complex with PPi bound (PDB:1S77) [17]; Right panel: The three-state MSM of the PPi release process derived from 100 × 20 ns MD simulations (by clustering ~10⁶ conformations into 200 microstates etc.) [31]. Note that the PPi group is depicted in red spheres, while the O-helix is colored green. (B) The schematics of an incomplete Brownian-ratchet device, which is still lack of the ‘pawl’. (C) The six-state MSM of the RNAP translocation on the DNA (129 × 80 ns all-atom MD simulation, clustering ~ 9 × 10⁵ conformations into 500 microstates etc.) [34]. The translocation starts after the PPi release from the product complex, or the pre-translocation state (S1), transiting all the way (via S2-S5 and mainly S3) to the post-translocation state (S6; PDB: 1MSW) [16] (populations and transition rates are labeled). Note that both the O-helix (green) and Y-helix (cyan) on the fingers subdomain are shown (with open/closed labeled), along with Y639 and F644 that are key residues in the translocation. The RNA and template DNA nucleotides are colored in blue and red, respectively. (D) The probability distributions of the rotational angles of the O-helix and the Y-helix during translocation process (from S1 to S6) are presented (as taken from [34]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Selective ratcheting of T7 RNAP on DNA as nucleotides are differentiated and selected as being incorporated to the growing end of RNA in synthesis. (A) A schematics showing free energy profiles of the cognate/right and non-cognate/wrong nucleotide addition cycle (NAC), which includes translocation, nucleotide binding/pre-insertion, nucleotide insertion, catalysis, and PPi product release. The nucleotide pre-insertion, insertion, and catalysis together serve as a ‘pawl’ for the ratchet. (B) The molecular views around the active site of the pre-insertion complexes modeled for our simulation studies [30,64]. Upper row: the non-cognate rGTP pre-insertion complexes, made on-path and off-path [64], respectively; Lower row: the cognate rATP and the non-cognate dATP (off-path) pre-insertion complexes [30]. (C) The free energy profiles or PMFs calculated from umbrella sampling simulations of the cognate rATP and non-cognate rGTP off-path insertion [36]. (D) The molecular views around the active site of T7 RNAP from representative snapshots captured in the umbrella sampling MD simulations of cognate rATP, non-cognate rGTP (off-path) and dATP (on-path) insertion process [36]. See text for further illustration.