RNA polymerase recycling by mycobacterial HelD: an alternative, HelD-protected pathway of transcription initiation

Tomáš Kovaľ1*, Nabajyoti Borah2,3*, Petra Sudzinová2, Barbora Brezovská2, Hana Šanderová2, Viola Vaňková Hausnerová2, Alena Křenková4, Martin Hubálek4, Mária Trundová1, Kristýna Adámková1, Jarmila Dušková1, Marek Schwarz2, Jana Wiedermannová2, Jan Dohnálek1#, Libor Krásný2# and Tomáš Kouba4#

1Institute of Biotechnology of the Czech Academy of Sciences, Průmyslová 595, 252 50 Vestec, Czech Republic

2Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic

3Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic

4Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 542/2, 160 00 Prague, Czech Republic

 

*These authors contributed equally

#email: tomas.kouba@uochb.cas.cz; krasny@biomed.cas.cz; dohnalek@ibt.cas.cz

 

Mycobacterial HelD is a transcription factor that binds stalled RNA polymerase (RNAP), dissociates it from nucleic acids and, if present, 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 solved a series of RNAP-σA-RbpA-HelD structures with or without 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. Taken together, HelD mediates an alternative pathway of transcription initiation where this process is protected from rifampicin until the last possible moment.