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

Bacterial RNA polymerases (RNAP) form distinct holoenzymes with different sigma factors to initiate diverse gene expression programs. In this study, we report a cryo-EM structure at 2.49 angstrom of RNA polymerase transcription complex containing a temperature-sensitive bacterial sigma factor, sigma(32) ( sigma(32)-RPo). The structure of sigma(32)-RPo reveals key interactions essential for the assembly of E. coli sigma(32)-RNAP holoenzyme and for promoter recognition and unwinding by sigma(32). Specifically, a weak interaction between sigma(32) and 35/-10 spacer is mediated by T128 and K130 in sigma(32). A histidine in sigma(32), rather than a tryptophan in sigma(70), acts as a wedge to separate the base pair at the upstream junction of the transcription bubble, highlighting the differential promoter-melting capability of different residue combinations. Structure superimposition revealed relatively different orientations between beta FTH and sigma(4) from other sigma-engaged RNAPs and biochemical data suggest that a biased sigma(4)-fiFTH configuration may be adopted to modulate binding affinity to promoter so as to orchestrate the recognition and regulation of different promoters. Collectively, these unique structural features advance our understanding of the mechanism of transcription initiation mediated by different sigma factors.