Utilization of variably spaced promoter‐like elements by the bacterial RNA polymerase holoenzyme during early elongation

Abstract

SUMMARY The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme in complex with a σ factor that is required for promoter-specific transcription initiation. During initiation, members of the σ70 family of σ factors contact two conserved promoter elements, the −10 and −35 elements, that are separated by ~17 base pairs (bp). σ70 family members contain four flexibly linked domains. Two of these domains, σ2 and σ4, contain determinants for interactions with the promoter −10 and −35 elements, respectively. σ2 and σ4 also contain core-binding determinants. When bound to core the inter-domain distance between σ2 and σ4 matches the distance between promoter elements separated by ~17 bp. Prior work indicates that during early elongation the nascent RNA-assisted displacement of σ4 from core can enable the holoenzyme to adopt a configuration in which σ2 and σ4 are bound to “promoter-like” DNA elements separated by a single base pair. Here we demonstrate that holoenzyme can also adopt configurations in which σ2 and σ4 are bound to “promoter-like” DNA elements separated by 0, 2 or 3 bp. Thus, our findings suggest that displacement of σ4 from core enables the RNAP holoenzyme to adopt a broad range of “elongation-specific” configurations.

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

“…This position is identical to the positions of upstream ExoIII barriers that were observed previously in σ 70 -containing promoter-proximal paused complexes (35,36). As opposed to the σ-less reactions, the patterns of upstream ExoIII digestions in σ 70 -containing TECs were identical for stalled 20, 23, 24-mer and paused 25-mer complexes (Figure 4A and C).…”

The reduction in sigma-promoter recognition flexibility as induced by core RNAP is required for sigma to discern the optimal promoter spacing

“…The relatively lower flexibility in simultaneous binding of the promoter − 10 and − 35 elements by SND100-σ A in the context of holo RNAP (Figure 2A, right-hand panel) is similar to that observed for E. coli σ 70 in recognition of the − 10-and − 35-like sequences in the λQ-engaged transcription elongation complex [40]. The ability of the DL mutants with various lengths of linker deletion to discern the optimal promoter spacing in the context of holo RNAP ( Figure 5B) indicates that sensing of the optimal promoter spacing by σ A is not directly controlled by the length of linker in σ A Region 3.2 instead it is a result of reduction in the flexibility of σ A -promoter recognition as induced by core RNAP.…”

Structure of the DNA-Binding and RNA-Polymerase-Binding Region of Transcription Antitermination Factor λQ

“…The TTGACT motif is located 1 bp upstream of the pause-inducing sequence, where σR2 is bound. Thus, the λQ-σR4 interaction stabilizes a non-standard conformation of the transcription complex in which σR2 and σR4 are bound to promoter −10-like and promoter −35-like DNA elements separated by a non-standard length spacer (1 bp spacer vs ~17 bp spacer; Figure 1B, C) (Devi et al, 2010; Nickels et al, 2002b). The interaction between λQ and σR4 stabilizes this non-standard conformation of RNAP in which σR4 is displaced from its normal position on the tip of the β flap and is relocated, by ~50 to 60 Å to the base of the β flap (Devi et al, 2010).…”