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

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
• adenosintrifosfát metabolismus   MeSH
• bakteriální proteiny * metabolismus   genetika   MeSH
• DNA řízené RNA-polymerasy * metabolismus   MeSH
• genetická transkripce MeSH
• iniciace genetické transkripce * MeSH
• Mycobacterium smegmatis * metabolismus   genetika   MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií MeSH
• rifampin * farmakologie   MeSH
• sigma faktor * metabolismus   genetika   MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• bakteriální proteiny * MeSH
• DNA řízené RNA-polymerasy * MeSH
• rifampin * 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

Bacteria employ small non-coding RNAs (sRNAs) to regulate gene expression. Ms1 is an sRNA that binds to the RNA polymerase (RNAP) core and affects the intracellular level of this essential enzyme. Ms1 is structurally related to 6S RNA that binds to a different form of RNAP, the holoenzyme bearing the primary sigma factor. 6S RNAs are widespread in the bacterial kingdom except for the industrially and medicinally important Actinobacteria. While Ms1 RNA was identified in Mycobacterium, it is not clear whether Ms1 RNA is present also in other Actinobacteria species. Here, using a computational search based on secondary structure similarities combined with a linguistic gene synteny approach, we identified Ms1 RNA in Streptomyces. In S. coelicolor, Ms1 RNA overlaps with the previously annotated scr3559 sRNA with an unknown function. We experimentally confirmed that Ms1 RNA/scr3559 associates with the RNAP core without the primary sigma factor HrdB in vivo. Subsequently, we applied the computational approach to other Actinobacteria and identified Ms1 RNA candidates in 824 Actinobacteria species, revealing Ms1 RNA as a widespread class of RNAP binding sRNAs, and demonstrating the ability of our multifactorial computational approach to identify weakly conserved sRNAs in evolutionarily distant genomes.

• Klíčová slova
• 6S RNA, Actinobacteria, Ms1 RNA, Mycobacterium, Streptomyces, gene synteny, sRNA,
• Publikační typ
• časopisecké články MeSH

Regulatory RNAs control a number of physiological processes in bacterial cells. Here we report on a 6S-like RNA transcript (scr3559) that affects both development and antibiotic production in Streptomyces coelicolor. Its expression is enhanced during the transition to stationary phase. Strains that over-expressed the scr3559 gene region exhibited a shortened exponential growth phase in comparison with a control strain; accelerated aerial mycelium formation and spore maturation; alongside an elevated production of actinorhodin and undecylprodigiosin. These observations were supported by LC-MS analyses of other produced metabolites, including: germicidins, desferrioxamines, and coelimycin. A subsequent microarray differential analysis revealed increased expression of genes associated with the described morphological and physiological changes. Structural and functional similarities between the scr3559 transcript and 6S RNA, and its possible employment in regulating secondary metabolite production are discussed.

• Klíčová slova
• 6S RNA, Streptomyces, antibiotics, secondary metabolism, small RNA,
• Publikační typ
• časopisecké články 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
• bakteriální proteiny chemie   metabolismus   ultrastruktura   MeSH
• DNA bakterií chemie   metabolismus   MeSH
• DNA řízené RNA-polymerasy chemie   metabolismus   ultrastruktura   MeSH
• elektronová kryomikroskopie MeSH
• katalytická doména MeSH
• molekulární modely MeSH
• Mycobacterium smegmatis enzymologie   MeSH
• nukleové kyseliny metabolismus   MeSH
• proteinové domény MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA řízené RNA-polymerasy MeSH
• nukleové kyseliny MeSH

Secondary data structure of RNA molecules provides insights into the identity and function of RNAs. With RNAs readily sequenced, the question of their structural characterization is increasingly important. However, RNA structure is difficult to acquire. Its experimental identification is extremely technically demanding, while computational prediction is not accurate enough, especially for large structures of long sequences. We address this difficult situation with rPredictorDB, a predictive database of RNA secondary structures that aims to form a middle ground between experimentally identified structures in PDB and predicted consensus secondary structures in Rfam. The database contains individual secondary structures predicted using a tool for template-based prediction of RNA secondary structure for the homologs of the RNA families with at least one homolog with experimentally solved structure. Experimentally identified structures are used as the structural templates and thus the prediction has higher reliability than de novo predictions in Rfam. The sequences are downloaded from public resources. So far rPredictorDB covers 7365 RNAs with their secondary structures. Plots of the secondary structures use the Traveler package for readable display of RNAs with long sequences and complex structures, such as ribosomal RNAs. The RNAs in the output of rPredictorDB are extensively annotated and can be viewed, browsed, searched and downloaded according to taxonomic, sequence and structure data. Additionally, structure of user-provided sequences can be predicted using the templates stored in rPredictorDB.

• MeSH
• databáze nukleových kyselin * MeSH
• konformace nukleové kyseliny * MeSH
• RNA * chemie   genetika   MeSH
• software * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA * MeSH

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.

• MeSH
• buněčné jádro genetika   MeSH
• Cajalova tělíska genetika   metabolismus   MeSH
• HeLa buňky MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U2 genetika   MeSH
• regulace genové exprese genetika   MeSH
• RNA malá jaderná genetika   MeSH
• spliceozomy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malý jaderný ribonukleoprotein U2 MeSH
• RNA malá jaderná MeSH
• U2 small nuclear RNA MeSH Prohlížeč