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.

• MeSH
• bakteriální proteiny chemie   metabolismus   ultrastruktura   MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   metabolismus   ultrastruktura   MeSH
• elektronová kryomikroskopie MeSH
• katalytická doména MeSH
• molekulární modely MeSH
• Mycobacterium smegmatis enzymologie   MeSH
• nukleové kyseliny metabolismus   MeSH
• proteinové domény MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• nukleové kyseliny MeSH

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

• Klíčová slova
• RNAP, RNase J1, stalling, torpedo, transcription-replication collision,
• MeSH
• Bacillus subtilis enzymologie   genetika   MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální RNA genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• exoribonukleasy metabolismus   MeSH
• genetická transkripce MeSH
• messenger RNA genetika   metabolismus   MeSH
• regulace genové exprese u bakterií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• DNA řízené RNA-polymerasy MeSH
• exoribonukleasy MeSH
• messenger RNA MeSH

Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σA) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σA. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σA. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.

• MeSH
• bakteriální chromozomy MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• malá nekódující RNA chemie   genetika   metabolismus   MeSH
• Mycobacterium smegmatis enzymologie   genetika   růst a vývoj   MeSH
• Mycobacterium genetika   MeSH
• sigma faktor metabolismus   MeSH
• syntenie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA řízené RNA-polymerasy MeSH
• malá nekódující RNA MeSH
• sigma faktor MeSH