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

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

• MeSH
• Bacillus subtilis genetics   metabolism   MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• RNA, Bacterial * metabolism   genetics   MeSH
• Corynebacterium glutamicum genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases * metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Nucleic Acid Conformation MeSH
• Mycobacterium smegmatis genetics   metabolism   enzymology   MeSH
• Mycobacterium tuberculosis genetics   metabolism   MeSH
• RNA, Untranslated MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Streptomyces coelicolor genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 6S RNA MeSH Browser
• Bacterial Proteins * MeSH
• RNA, Bacterial * MeSH
• DNA-Directed RNA Polymerases * MeSH
• RNA, Untranslated MeSH
• Sigma Factor * 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