Distinct functions of the RNA polymerase σ subunit region 3.2 in RNA priming and promoter escape

Abstract

The σ subunit of bacterial RNA polymerase (RNAP) has been implicated in all steps of transcription initiation, including promoter recognition and opening, priming of RNA synthesis, abortive initiation and promoter escape. The post-promoter-recognition σ functions were proposed to depend on its conserved region σ3.2 that directly contacts promoter DNA immediately upstream of the RNAP active centre and occupies the RNA exit path. Analysis of the transcription effects of substitutions and deletions in this region in Escherichia coli σ70 subunit, performed in this work, suggests that (i) individual residues in the σ3.2 finger collectively contribute to RNA priming by RNAP, likely by the positioning of the template DNA strand in the active centre, but are not critical to promoter escape; (ii) the physical presence of σ3.2 in the RNA exit channel is important for promoter escape; (iii) σ3.2 promotes σ dissociation during initiation and suppresses σ-dependent promoter-proximal pausing; (iv) σ3.2 contributes to allosteric inhibition of the initiating NTP binding by rifamycins. Thus, region σ3.2 performs distinct functions in transcription initiation and its inhibition by antibiotics. The B-reader element of eukaryotic factor TFIIB likely plays similar roles in RNAPII transcription, revealing common principles in transcription initiation in various domains of life.