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.

• Klíčová slova
• MoaB2, RNA polymerase, mycobacteria, transcription, σA,
• MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy metabolismus   genetika   MeSH
• genetická transkripce MeSH
• krystalografie rentgenová MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• regulace genové exprese u bakterií * MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory * metabolismus   genetika   MeSH
• vazba proteinů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy MeSH
• sigma faktor * MeSH
• transkripční faktory * 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 genetika   metabolismus   MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• bakteriální RNA * metabolismus   genetika   MeSH
• Corynebacterium glutamicum genetika   metabolismus   MeSH
• DNA řízené RNA-polymerasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• konformace nukleové kyseliny MeSH
• Mycobacterium smegmatis genetika   metabolismus   enzymologie   MeSH
• Mycobacterium tuberculosis genetika   metabolismus   MeSH
• nekódující RNA MeSH
• regulace genové exprese u bakterií MeSH
• sigma faktor * metabolismus   genetika   MeSH
• Streptomyces coelicolor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 6S RNA MeSH Prohlížeč
• bakteriální proteiny * MeSH
• bakteriální RNA * MeSH
• DNA řízené RNA-polymerasy * MeSH
• nekódující RNA MeSH
• sigma faktor * 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.

• Klíčová slova
• HelD/HelR, RNA polymerase, antibiotics, bacteria, resistance, rifampicin,
• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria * genetika   metabolismus   MeSH
• bakteriální léková rezistence MeSH
• DNA řízené RNA-polymerasy genetika   metabolismus   MeSH
• DNA MeSH
• rifampin * farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• DNA řízené RNA-polymerasy MeSH
• DNA MeSH
• rifampin * MeSH