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

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.

• Klíčová slova
• Bacillus, RNA polymerase, molecular modeling, nuclear magnetic resonance (NMR), protein structure, transcription initiation factor,
• MeSH
• Bacillus subtilis metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• molekulární modely * MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• sigma faktor chemie   genetika   metabolismus   MeSH
• stabilita proteinů MeSH
• strukturní homologie proteinů MeSH
• Thermotoga maritima enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• izotopy dusíku MeSH
• izotopy uhlíku MeSH
• peptidové fragmenty MeSH
• podjednotky proteinů MeSH
• rekombinantní proteiny MeSH
• sigma faktor MeSH