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

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP.IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

• Keywords
• RNA polymerase, bacterial transcription, conformational change, cryo-electron microscopy, mycobacteria, protein structure, transcription initiation factor,
• MeSH
• DNA-Directed RNA Polymerases chemistry   ultrastructure   MeSH
• Cryoelectron Microscopy MeSH
• Holoenzymes chemistry   ultrastructure   MeSH
• Protein Conformation MeSH
• Mycobacterium smegmatis enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• Holoenzymes MeSH