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
• Bacterial Proteins chemistry   metabolism   ultrastructure   MeSH
• DNA, Bacterial chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   metabolism   ultrastructure   MeSH
• Cryoelectron Microscopy MeSH
• Catalytic Domain MeSH
• Models, Molecular MeSH
• Mycobacterium smegmatis enzymology   MeSH
• Nucleic Acids metabolism   MeSH
• Protein Domains MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Nucleic Acids 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.

• Keywords
• RNAP, RNase J1, stalling, torpedo, transcription-replication collision,
• MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Bacterial Proteins metabolism   MeSH
• RNA, Bacterial genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Exoribonucleases metabolism   MeSH
• Transcription, Genetic MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• RNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Exoribonucleases MeSH
• RNA, Messenger 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.

• Keywords
• Bacillus, RNA polymerase, molecular modeling, nuclear magnetic resonance (NMR), protein structure, transcription initiation factor,
• MeSH
• Bacillus subtilis metabolism   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• DNA, Bacterial chemistry   metabolism   MeSH
• DNA-Directed RNA Polymerases chemistry   genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Nitrogen Isotopes MeSH
• Carbon Isotopes MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Conserved Sequence MeSH
• Models, Molecular * MeSH
• Peptide Fragments chemistry   genetics   metabolism   MeSH
• Protein Subunits MeSH
• Recombinant Proteins chemistry   metabolism   MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Sigma Factor chemistry   genetics   metabolism   MeSH
• Protein Stability MeSH
• Structural Homology, Protein MeSH
• Thermotoga maritima enzymology   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Bacterial Proteins MeSH
• DNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Nitrogen Isotopes MeSH
• Carbon Isotopes MeSH
• Peptide Fragments MeSH
• Protein Subunits MeSH
• Recombinant Proteins MeSH
• Sigma Factor MeSH