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.

• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases * metabolism   MeSH
• Transcription, Genetic MeSH
• Transcription Initiation, Genetic * MeSH
• Mycobacterium smegmatis * metabolism   genetics   MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Rifampin * pharmacology   MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Transcription Factors metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Bacterial Proteins * MeSH
• DNA-Directed RNA Polymerases * MeSH
• Rifampin * MeSH
• Sigma Factor * MeSH
• Transcription Factors MeSH

Rifampicin is a clinically important antibiotic that binds to, and blocks the DNA/RNA channel of bacterial RNA polymerase (RNAP). Stalled, nonfunctional RNAPs can be removed from DNA by HelD proteins; this is important for maintenance of genome integrity. Recently, it was reported that HelD proteins from high G+C Actinobacteria, called HelR, are able to dissociate rifampicin-stalled RNAPs from DNA and provide rifampicin resistance. This is achieved by the ability of HelR proteins to dissociate rifampicin from RNAP. The HelR-mediated mechanism of rifampicin resistance is discussed here, and the roles of HelD/HelR in the transcriptional cycle are outlined. Moreover, the possibility that the structurally similar HelD proteins from low G+C Firmicutes may be also involved in rifampicin resistance is explored. Finally, the discovery of the involvement of HelR in rifampicin resistance provides a blueprint for analogous studies to reveal novel mechanisms of bacterial antibiotic resistance.

• Keywords
• HelD/HelR, RNA polymerase, antibiotics, bacteria, resistance, rifampicin,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteria * genetics   metabolism   MeSH
• Drug Resistance, Bacterial MeSH
• DNA-Directed RNA Polymerases genetics   metabolism   MeSH
• DNA MeSH
• Rifampin * pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH
• Rifampin * MeSH

During the first step of gene expression, RNA polymerase (RNAP) engages DNA to transcribe RNA, forming highly stable complexes. These complexes need to be dissociated at the end of transcription units or when RNAP stalls during elongation and becomes an obstacle ('sitting duck') to further transcription or replication. In this review, we first outline the mechanisms involved in these processes. Then, we explore in detail the torpedo mechanism whereby a 5'-3' RNA exonuclease (torpedo) latches itself onto the 5' end of RNA protruding from RNAP, degrades it and upon contact with RNAP, induces dissociation of the complex. This mechanism, originally described in Eukaryotes and executed by Xrn-type 5'-3' exonucleases, was recently found in Bacteria and Archaea, mediated by β-CASP family exonucleases. We discuss the mechanistic aspects of this process across the three kingdoms of life and conclude that 5'-3' exoribonucleases (β-CASP and Xrn families) involved in the ancient torpedo mechanism have emerged at least twice during evolution.

• MeSH
• Archaea genetics   MeSH
• Bacteria genetics   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA metabolism   MeSH
• Eukaryota genetics   MeSH
• Exoribonucleases metabolism   MeSH
• Transcription, Genetic MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• 5'-exoribonuclease MeSH Browser
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH
• Exoribonucleases MeSH

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase-α, β, β'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the β or β' subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed a severe decrease in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

• MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Genes, Bacterial MeSH
• Gene Deletion MeSH
• DNA-Directed RNA Polymerases chemistry   genetics   metabolism   MeSH
• Gene Duplication MeSH
• Endoribonucleases genetics   physiology   MeSH
• Transcription, Genetic MeSH
• Homeostasis MeSH
• RNA, Messenger metabolism   MeSH
• Evolution, Molecular MeSH
• Mutation MeSH
• Suppression, Genetic MeSH
• Transcriptome MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Endoribonucleases MeSH
• RNA, Messenger MeSH

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

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

• MeSH
• Bacillus subtilis enzymology   MeSH
• Deoxyribonucleotides biosynthesis   chemistry   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Escherichia coli enzymology   MeSH
• Transcription, Genetic * MeSH
• Templates, Genetic MeSH
• Nucleic Acid Conformation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deoxyribonucleotides MeSH
• DNA-Directed RNA Polymerases MeSH
• DNA MeSH

RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A+T Firmicutes phylum of Gram-positive bacteria and have named it ε. Previously ε had been identified as a small protein (ω1) that copurified with RNA polymerase. We have solved the structure of ε by X-ray crystallography and show that it is not an ω subunit. Rather, ε bears remarkable similarity to the Gp2 family of phage proteins involved in the inhibition of host cell transcription following infection. Deletion of ε shows no phenotype and has no effect on the transcriptional profile of the cell. Determination of the location of ε within the assembly of RNA polymerase core by single-particle analysis suggests that it binds toward the downstream side of the DNA binding cleft. Due to the structural similarity of ε with Gp2 and the fact they bind similar regions of RNA polymerase, we hypothesize that ε may serve a role in protection from phage infection.

• MeSH
• Bacillus subtilis enzymology   MeSH
• DNA-Directed RNA Polymerases chemistry   genetics   metabolism   MeSH
• Phylogeny MeSH
• Protein Conformation MeSH
• Models, Molecular MeSH
• Molecular Sequence Data MeSH
• Protein Subunits MeSH
• Gene Expression Regulation, Enzymologic MeSH
• Gene Expression Regulation, Bacterial MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Protein Subunits MeSH