A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli

Abstract

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli. Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage

“…21,22,[29][30][31][32][33] Our results show further that backtrack-recovery is mediated by intrinsic RNA cleavage and not not by diffusional Brownian motion, indicating that a subset of paused TECs assume a conformational state in which intrinsic and GreBassisted RNA cleavage is hindered, delaying as such the escape to productive elongation. 25,34,40,[42][43][44][45][46] The availability of large datasets also allowed us to interrogate the sources of the poorly understood heterogeneity in transcription velocity and pause dynamics, providing support for previously postulated state-switching that we could link to stochastic alterations in the frequency of short pauses. 24,26,29,32,[47][48][49][50] We present a unified mechanistic model that integrates all key findings of previous biochemical and singlemolecule studies, and describes the origin and hierarchy of intrinsic pause states as framework for future studies.…”

“…Our observation that GreA does not affect the apparent RNAp pause dynamics can be understood by the fact that GreA mainly affects short backtracks of ≤3 nucleotides. 34,40,42,45,69,70 This result indicates that short backtracks are short-lived and either recover quickly (≤1 s) via cleavage or diffusion, or lead to extended backtracking. This scenario could explain why short backtracks remain undetected in our analysis, as their contribution to the DWT distribution might be overshadowed by the elemental pause (lifetime ~ 1 s), as proposed by Saba et al 21 Furthermore, if short backtracks are predominantly recovered by diffusion, which we can neither confirm nor exclude from our data, the GreA-mediated cleavage might not be able to substantially suppress the occurrence of deeper backtracks.…”

“…The RNAp can recover from backtracking either by diffusing forward, placing the 3'end of the transcript in the active site, or by cleaving the backtracked RNA, thereby freeing the active site. 40,43,45,[66][67][68] Previous single-molecule studies have suggested that diffusion provides the dominant recovery mechanism. 15,23,36,[38][39][40][41] Our data contradicts this view: we observe only a limited degree of force dependency for kP2, and the lifetimes of P2 and P3 states do not seem to be affected by the external force,.…”

“…The prokaryotic Gre factors augment the intrinsic cleavage rate of RNAp, promoting cleavage-mediated recovery of backtracked TEC. 25,34,40,42,43,45,66 To increase the measurable effect of Gre factors, we applied an OF that favors the long-lived pauses (Figure 4; Figure S5). Figure 5A shows the effect of 2 µM GreA and 2 µM GreB have on the DWT distribution measured at 9 pN OF, 1 mM [NTP].…”

RNA polymerase active centre compensates for the absence of transcription proofreading factors in Cyanobacteria

“…Acidic amino acid residues at the tip of the coiled-coil domain of GreA stabilise the second Mg 2+ ion and coordinate a water molecule greatly increasing the efficiency of hydrolysis (18)(19)(20). As a result GreA improves the fidelity of transcription by up to two orders of magnitude in some instances (21). Gre factors can also reactivate correctly paired complexes that have backtracked and arrested for different reasons (such as prolonged pauses or DNA lesions) (22,23).…”