In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

• Keywords
• MoaB2, RNA polymerase, mycobacteria, transcription, σA,
• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• DNA-Directed RNA Polymerases metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Crystallography, X-Ray MeSH
• Mycobacterium smegmatis * metabolism   genetics   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Transcription Factors * metabolism   genetics   MeSH
• Protein Binding * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• DNA-Directed RNA Polymerases MeSH
• Sigma Factor * MeSH
• Transcription Factors * MeSH

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

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