Region 4 of the RNA polymerase σ subunit counteracts pausing during initial transcription

Abstract

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The antipausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This “RNAP boosting” activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

Structural Insight into the Mechanism of σ32-Mediated Transcription Initiation of Bacterial RNA Polymerase

“…Our structure shows that the C:G bases at position −12 are paired, while the A:T bases at position −11 are unwound (Figure 1F). This observation strongly suggests that σ 2 unwinds promoter DNA at the −11/−12 junction, which is consistent with that of σ 70 [49,50]. Specifically, H107 blocks the path upstream dsDNA and serves as a wedge to disrupt base stacking between positions A −11 and C −12 (Figure 1F).…”

“…These results indicated the importance of σ 4 -βFTH interaction in σ 32 -promoter recognition and also highlighted the important role of βFTH in orchestrating the recognition of other σ promoters. Although our study shows that the σ 4 -βFTH interaction affects transcription activity by influencing the binding affinity of σ 4 to the promoter, it seems that other steps such as promoter escape and transcription pausing may also play a role [16,49]. Nevertheless, our study indicates that the σ 4 -βFTH interaction is tuned to balance the distinct transcription efficiencies of different σ factors.…”

“…σ 70 , σ 38 , and σ 28 of E. coli unwind promoter DNA at the −11/−12 junction [26,44,49], while σ 24 unwinds promoter DNA at a position 1 bp upstream of the transcription bubble [15]. Our structure shows that the C:G bases at position −12 are paired, while the A:T bases at position −11 are unwound (Figure 1F).…”

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

“…In this study we show that Mtb RNAP harboring the group II σ Β factor can spontaneously oligomerize into symmetric octamers in which RNAP is captured in an inactive conformation with the domain σ4 unloaded from the RNAP core. RNAP oligomerization is reversed by the global transcriptional activator RbpA that, according to smFRET and biochemical studies 6 , 44 induces σ4 domain loading onto RNAP (see model in Fig. 7 ).…”

Structural basis of the mycobacterial stress-response RNA polymerase auto-inhibition via oligomerization

“…In this study we show that Mtb RNAP harboring the group II s B factor can spontaneously oligomerize into symmetric octamers in which RNAP is captured in an inactive conformation with the domain s4 unloaded from the RNAP core. RNAP oligomerization is reversed by the global transcriptional activator RbpA that, according to smFRET and biochemical studies (Brodolin and Morichaud, 2021;Vishwakarma et al, 2018), induces s4 domain loading onto RNAP (see model in Figure 7). In bacterial cells, s factors compete for a general RNAP core pool (Grigorova et al, 2006).…”