Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 128 44 Prague Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences Průmyslová 595 252 50 Vestec Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo náměstí 542 2 160 00 Prague Czech Republic

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