Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP.IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

Department of Genetics and Microbiology Faculty of Science Charles University Prague Prague Czech Republic

Division of Biomolecular Physics Institute of Physics Faculty of Mathematics and Physics Charles University Prague Prague Czech Republic

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic

MRC Laboratory of Molecular Biology Cambridge United Kingdom

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MoaB2, a newly identified transcription factor, binds to σA in Mycobacterium smegmatis

Mycobacterial HelD connects RNA polymerase recycling with transcription initiation

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase