Structures and mechanism of transcription initiation by bacterial ECF factors

Abstract

Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE—the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

Rewiring the specificity of extra-cytoplasmic function sigma factors

“…We calculated the MI between all positions in the promoter and protein alignments and found that only a few amino acids -nucleotide positions had high MI ( Figure 1B). High MI amino acid -nucleotide interactions were consistent with specificity determining interactions observed in the structures of 3 divergent ECF σ s bound to their cognate promoters: E. coli RpoE and Mycobacterium tuberculosis SigH and SigL (Lane and Darst 2006;Campagne et al 2014;Lin et al 2019;Fang et al 2019;Li et al 2019; Figure 1B). Taken together, these data suggest that the mechanism of ECF σ -promoter interaction is conserved in a majority of ECF σ s despite their extensive sequence divergence, implying that our collection of matching ECF σ s and putative promoters can serve as a dictionary in which the promoter specificity conferred by any amino acid at key DNA recognizing positions can be determined.…”

“…Since the amino acid at Arg173 RpoE is not positioned to make base-specific interactions, this is likely due to a role in positioning the amino acid at Finally, we tested the effect of amino acid substitutions at the Asn84 RpoE position of ECF11_987 on -12 and -13 nucleotide specificity ( Supplementary Table 3). Whereas all previously solved DNA-bound ECF σ structures reported that the amino acid at the Asn84 RpoE position makes a hydrogen bond with the -12 base of the promoter (Campagne et al 2014;Lin et al 2019;Fang et al 2019;Li et al 2019), our MI analysis suggested that the Asn84 RpoE identity determines both -12 and -13 nucleotide specificity. We therefore tested the ability of all Asn84 RpoE substitutions of ECF11_987 to drive transcription from 16 variants of the ECF11_987 promoter modified at the -13 and/or -12 nucleotide positions (Methods).…”

“…We similarly tested the effect of substitutions at the Ser172 RpoE position on -34 specificity using ECF14_1324 and its cognate promoter pECF14 (Rhodius et al 2013;Methods; Supplementary Table 3). DNA bound ECF σ structures (Lane and Darst 2006;Fang et al 2019;Li et al 2019) and our MI analysis suggest that -34 is recognized through a multipartite interaction driven primarily by the amino acid at the Ser172 RpoE position but with contributions from the amino acids at the Gly168 RpoE and Thr169 RpoE positions. Consistent with multipartite determination of -34 specificity, we found that only 4/20 Ser172 RpoE substitutions were active.…”

RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription

“…2E). We interpret the successive changes in [6][7][8]. As a first step to assess whether the difference in s-finger length and sequence for ECF s factors vs. primary s factors affects the mechanism of initiation-factor displacement, we have performed an analysis analogous to that in Fig.…”

“…S4A-B) Stepwise, RNA-driven displacement of the s finger: ECF s factor s L The s fingers of different ECF s factors vary in length and sequence (ref. 6). As a second step to assess whether the difference in s-finger length and sequence for ECF s factors vs. primary s factors affects the mechanism of initiation-factor displacement, we have performed an analysis analogous to that in Fig.…”