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In bacteria, σ28 is the flagella‐specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella‐related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28‐dependent promoter recognition remains uncharacterized. Here, we report cryo‐EM structures of E. coli σ28‐dependent transcribing complexes on a complete flagella‐specific promoter. These structures reveal how σ28‐RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc‐binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non‐template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed‐to‐open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/−35 element interactions. Synopsis: Cryo‐EM structures of a σ28‐transcription initiation complex (TIC) reveal a novel role of β′ bacterial RNA polymerase (RNAP) zinc‐binding domain (ZBD) in transcription initiation. Relocation of the ZBD may compensate for weak σ4/−35 element interactions and hereby enhance transcription from this type of promoters. Cryo‐EM reconstructions of σ28‐TIC at 3.86 Å (state 1) and 3.91 Å (state 2) define the specific promoter recognition by σ28.Cryo‐EM structure of state‐2 σ28‐TIC reveals a distinct architecture of β′ zinc binding domain of RNAP.ZBD relocation has an overall effect of facilitating the isomerization ofRNAP‐promoter closed to open complex to enhance transcription.ZBD relocation could be exploited by the σ70‐family factors to enhanceTranscription by compensating for the weak interaction between σ4 and promoter −35 element. [ABSTRACT FROM AUTHOR]

Abstract: The baculovirus gene, protein kinase-I (pk-1) encodes a serine/threonine kinase that is essential for very late gene expression. Late and very late genes of the baculoviruses are transcribed by an α-amanitin resistant RNA polymerase. The very late gene promoter transcription initiation complex was isolated from nuclei of Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected Sf9 cells by DNA affinity purification and found to contain 4 major polypeptides of sizes ∼102, 38, 32, and 18kDa. The 32kDa polypeptide was immunoreactive to AcMNPV anti-pk-1 antibody and phosphorylated the 102kDa polypeptide, earlier reported as late gene expression factor LEF-8. Electrophoretic mobility shift assays with anti-pk-1 antibody indicated the binding of promoter DNA with recombinant AcMNPV-pk-1 and transcription initiation complex proteins. All these results suggested AcMNPV-pk-1 to be a component of the viral very late gene transcription initiation complex. [Copyright &y& Elsevier]