RESUMO
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.
Assuntos
Proteínas de Bactérias , RNA Polimerases Dirigidas por DNA , Mycobacterium smegmatis , Regiões Promotoras Genéticas , Rifampina , Fator sigma , Iniciação da Transcrição Genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Rifampina/farmacologia , Fator sigma/metabolismo , Fator sigma/genética , Fatores de Transcrição/metabolismo , Trifosfato de Adenosina/metabolismo , Transcrição Gênica , Regulação Bacteriana da Expressão Gênica , Ligação ProteicaRESUMO
RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.