Structural transitions in the transcription elongation complexes of bacterial RNA polymerase during σ-dependent pausing

Abstract

A transcription initiation factor, the σ70 subunit of Escherichia coli RNA polymerase (RNAP) induces transcription pausing through the binding to a promoter-like pause-inducing sequence in the DNA template during transcription elongation. Here, we investigated the mechanism of σ-dependent pausing using reconstituted transcription elongation complexes which allowed highly efficient and precisely controlled pause formation. We demonstrated that, following engagement of the σ subunit to the pause site, RNAP continues RNA synthesis leading to formation of stressed elongation complexes, in which the nascent RNA remains resistant to Gre-induced cleavage while the transcription bubble and RNAP footprint on the DNA template extend in downstream direction, likely accompanied by DNA scrunching. The stressed complexes can then either break σ-mediated contacts and continue elongation or isomerize to a backtracked conformation. Suppressing of the RNAP backtracking decreases pausing and increases productive elongation. On the contrary, core RNAP mutations that impair RNAP interactions with the downstream part of the DNA template stimulate pausing, presumably by destabilizing the stressed complexes. We propose that interplay between DNA scrunching and RNAP backtracking may have an essential role in transcription pausing and its regulation in various systems.

Gfh factors and NusA cooperate to stimulate transcriptional pausing and termination

“…We tested the effects of Dra Gfh1 (which has stronger effects on transcription than Gfh2, ) and NusA factors on different types of transcriptional pauses including (Fig. S1): (a) pretranslocated hairpin‐dependent hisP pause; (b) elemental pause ; (c) post‐translocated T7A1P pause ; and (d) backtracking‐dependent pause that constitutes a part of σ‐dependent pause signal found in the initially transcribed region of the lac UV5 promoter ( lac UV5P) .…”

“…Some DNA sequences can greatly increase the incidence of backtracking, resulting in RNAP pausing or permanent transcription arrest [10,11,17]. In particular, backtrack-inducing sequences were shown to contribute to r-dependent pausing, by determining the extent of RNA synthesis after recognition of a promoter-like motif by the r subunit and provoking RNAP backtracking to the relaxed paused state [26,33]. As we showed previously, a variant of the lacUV5 backtrack-inducing sequence (lacUV5P, Fig.…”

Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

“…To directly compare the pause-inducing properties of wild-type and mutant σs in the absence of the promoter escape step, we analyzed σ-dependent pausing in a model elongation complex assembled on a synthetic nucleic acid scaffold containing a −10-like pause-inducing signal optimally positioned for σ binding in respect to the RNA 3′-end (Supplementary Figure S3A) (23). The σ subunits were added at varying concentrations to pre-assembled elongation complexes, followed by nucleotide addition.…”

E. coli 6S RNA release from RNA polymerase requires σ70 ejection by scrunching and is orchestrated by a conserved RNA hairpin

“…The scrunched polymerase triggers a global rearrangement of the complex into a processive structure. This relatively poorly understood process often, but not always, results in the profound weakening of s 70 binding (Mukhopadhyay et al 2001;Zhilina et al 2012), which can in turn be recycled for subsequent rounds of transcription.…”