Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue

Abstract

The secondary channel (SC) of multisubunit RNA polymerases (RNAPs) allows access to the active site and is a nexus for the regulation of transcription. Multiple regulatory proteins bind in the SC and reprogram the catalytic activity of RNAP, but the dynamics of these factors' interactions with RNAP and how they function without cross-interference are unclear. In Escherichia coli, GreB is an SC protein that promotes proofreading by transcript cleavage in elongation complexes backtracked by nucleotide misincorporation. Using multiwavelength single-molecule fluorescence microscopy, we observed the dynamics of GreB interactions with elongation complexes. GreB binds to actively elongating complexes at nearly diffusion-limited rates but remains bound for only 0.3-0.5 s, longer than the duration of the nucleotide addition cycle but far shorter than the time needed to synthesize a complete mRNA. Bound GreB inhibits transcript elongation only partially. To test whether GreB preferentially binds backtracked complexes, we reconstituted complexes stabilized in backtracked and nonbacktracked configurations. By verifying the functional state of each molecular complex studied, we could exclude models in which GreB is selectively recruited to backtracked complexes or is ejected from RNAP by catalytic turnover. Instead, GreB binds rapidly and randomly to elongation complexes, patrolling for those requiring nucleolytic rescue, and its short residence time minimizes RNAP inhibition. The results suggest a general mechanism by which SC factors may cooperate to regulate RNAP while minimizing mutual interference.secondary channel | total internal reflection fluorescence | transcription elongation | backtracking T he multisubunit RNA polymerases (RNAPs) that synthesize mRNAs have catalytic subunits that are conserved across all domains of life but incorporate different regulatory structures and mechanisms (1, 2). Despite this divergence, all RNAPs have in common certain key target sites by which they interact with regulatory proteins. One of the most important of these is the secondary channel (SC), a pore extending from the outside surface of RNAP to the enzyme active site deeply buried in the center of the transcription elongation complex (EC) (3).Even within an individual bacterial species, multiple regulatory factors may interact with the SC. Escherichia coli has at least five proteins that are thought to bind in the RNAP SC to regulate transcription (4). These proteins have similar overall structures (5) but have qualitatively different effects on transcription complexes and thus have distinct roles in regulation.A well-characterized role of some SC proteins is the endonucleolytic rescue of backtracked ECs (6-8). In response to particular protein factors, template DNA sequences, transcript sequences, or nucleotide deprivation, the EC may undergo "backtracking" in which it reverse threads the nascent RNA and the DNA template by one or more base pair (9-12). In a backtracked conformation the EC is still intact, but the active site i...

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

“…Since GreB is known to facilitate recovery from deep backtracks, the observed acceleration of the exit rate ke2 is a clear indication that the P2 pause originates from backtracked TECs. 25,34,40,[42][43][44]66 The concomitant observed reduction in the occurrence of the P3 pause state, reflected by a decrease in the appearance of pauses exceeding 10 s in Figure 5A, supports our assumption that the entry to P3 competes with recovery from P2, implying that P3 may also originate from a backtrack-induced pause. Backtrack depth analysis reveals that P2 and P3 are associated with backtrack depths >4 nt (Figure S7).…”

“…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.…”

“…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].…”

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

“…Additional studies will be needed to determine the TraR concentration and its physiological consequences when TraR is made from the Py promoter and DksA is present. Whatever the TraR concentration is during conjugation, we note that single-molecule studies suggest that the short residence time of secondary channel binding factors on RNAP helps them cooperate to regulate transcription while minimizing mutual interference (38).…”