Altering the interaction between σ ⁷⁰ and RNA polymerase generates complexes with distinct transcription-elongation properties

Abstract

We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from 70 and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between 70 and RNA polymerase. Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of 70 during elongation, or by differential presence of elongation factors. We suggest that interactions between RNA polymerase and 70 may influence the ensemble of conformational states adopted by RNA polymerase during initiation. These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase.A ll multisubunit RNA polymerases use initiation factors to recognize their promoters, a strategy that allows tight base-specific binding during the initiation phase of transcription and nonspecific binding during elongation, after release of the initiation factor. This function is performed by in eubacterial cells (1). Almost all bacteria contain multiple factors, one directing transcription to housekeeping genes and the remainder directing transcription to genes encoding specialized functions (1).Genetic, biochemical, and structural characterization of the interaction between and RNA polymerase (E) (2-4) reveals that the interface between the two proteins is both extensive, having at least four regions of interaction (5-9), and dynamic, with some interactions depending on the formation of the preceding ones (5). Conformational changes in both partners result from this interaction. The changes in unmask and reposition its DNA-binding domains to allow promoter recognition (6,7,(10)(11)(12)(13)(14). Conformational changes in RNA polymerase may reposition portions of RNA polymerase in close contact with the nucleic acids, but the functional consequences of such changes are unknown.Usually, factors dissociate from elongating RNA polymerase shortly after RNA polymerase leaves the promoter (15-18) but remain associated with RNA polymerase longer than normal at a special class of promoters (19-21). The predominant eubacterial promoter has two conserved recognition sequences, centered at Ϫ10 and Ϫ35 bp upstream of the starting point of transcription (ϩ1). Promoters with a reiterated Ϫ10 motif in the initially transcribed region exhibit prolonged association. This motif was discovered first in promoters directing lambdoid phage late transcription. The recognition of the reiterated Ϫ10 region induces a transcription pause (19-23) that is required to load the Q elongation factor that antiterminates transcription (24). Reiterated Ϫ10 regions were identified recently (21-23) in a subset of bacterial promoters, including lacUV5. It is thought that dissociates shortly after passing the reiterated Ϫ10 region (23). E 70 from stationary cells may be refractory to dissociation as a significant fraction (Ϸ30%) of RNA polymerase purified from suc...

Single-Molecule Studies of RNA Polymerase: One Singular Sensation, Every Little Step It Takes

“…However, an alternative explanation is that RNAP molecules with otherwise identical primary structures might fold in different conformations with distinct transcriptional activities. This possibility has been advanced in reports of ensemble experiments (Berghofer-Hochheimer et al, 2005), single-molecule experiments (Davenport et al, 2000), and on theoretical grounds (Harrington et al, 2001). Moreover, molecules with similar tertiary structures may be subject to dynamic “state-switching” during elongation, adopting an activity state that persists throughout the transcription of an entire gene (Herbert et al, 2006; Neuman et al, 2003) or that rapidly inter-converts between activity states, possibly at each new template position.…”

A pathway branching in transcription initiation in Escherichia coli

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