Abstract
Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.
Highlights
During the first step in gene expression, transcription by RNA polymerase (RNAP) at ~30 nt/s or faster is interrupted by !1 s pause events every 100–200 bp (Landick, 2006; Larson et al, 2014; Chen et al, 2015)
To probe the elemental pause mechanism, we used kinetic analyses of elongating transcription complex (EC) reconstituted on a synthetic RNA-DNA scaffold encoding a consensus elemental pause sequence (Figure 1C and Figure 1—figure supplement 1A; Larson et al, 2014)
The elemental pause is an offline state that forms in competition with rapid elongation; the pause mechanism requires distinct EC and pause conformations rather than a single state with an energetic barrier to translocation
Summary
During the first step in gene expression, transcription by RNA polymerase (RNAP) at ~30 nt/s or faster is interrupted by !1 s pause events every 100–200 bp (Landick, 2006; Larson et al, 2014; Chen et al, 2015). Uncertainty exists as to whether the elemental pause is non-backtracked (Landick, 2006; Herbert et al, 2006; Kireeva and Kashlev, 2009; Kang et al, 2018a) or must be backtracked one or more registers (Dangkulwanich et al, 2013; Forde et al, 2002; Galburt et al, 2007; Mejia et al, 2015; Tadigotla et al, 2006; O Maoileidigh et al, 2011) To address these questions, we combined kinetic analyses of pausing using elemental pause sequence variants or mutant RNAPs with precise structural probes of translocation, trigger-loop folding, and clamp conformation. Our results lead us to propose a multistate model of elemental pausing in which template-base loading in a half-translocated offline intermediate limits pause escape
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