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| (A) Ms1 homologs were identified in Actinobacteria orders (in italics) or families (narrow italics) labeled by red. Actinobacteria groups with no identified Ms1 homologs and Ms1 flanking genes are in black, groups with identified Ms1 flanking genes but no Ms1 homologs in grey. The tree was adopted from Nouioui et al. (2018). (B) Genome sizes of the Corynebacteriales and (C) Actinobacteria. Only NCBI reference genomes are shown, number of genomes is indicated in brackets.
Source publication
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...
Citations
... In mycobacteria, no 6S RNA but Ms1 sRNA is found. Ms1, unlike 6S RNA, binds to the RNAP core and not the primary σ factor-containing holoenzyme (67,73). Deletion of the Ms1-encoding gene affects the levels of RNAP (74). ...
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.
IMPORTANCE
Mycobacteria 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.
... Recently, putative Ms1 RNAs homologs have been found among many actinobacteria, for example in Streptomyces coelicolor ( 36 ,37 ). In some actinobacteria, such as corynebacteria, neither 6S RNA nor Ms1 have been discovered so far ( 37 ) raising the question whether any similar RNA exists in these species. It is also unknown if both Ms1 and 6S RNA are present in Streptomyces coelicolor. ...
... 5´biotinylated oligonucleotide probes ( Supplementary Data ) were hybridized to the membrane and detected with the BrightStar BioDetect Kit (Ambion) or Novex or Tropix CDP STAR substrate (Ther-moFischer Scientific, Applied Biosystems) according to the manufacturer's instructions. 5´RACE and 3´RACE were performed according to the protocol used previously ( 37 ). ...
... Therefore, we also focused on RNAs associated with the transcriptional machinery in S. coelicolor A3(2) hrdB-HA, a strain in which the primary σ factor (HrdB) is endogenously tagged with hemagglutinin (HA) ( 38 ). We had previously shown that the anti-HA antibody immunoprecipitated the primary σ factor holoenzyme (HrdB-HA in complex with RNAP) ( 37 ). Here, we performed RNAP and HrdB-HA RIP-seq from exponential and stationary phases of growth (42 h and 66 h after germination, respectively, We confirmed that scr3559 sRNA is an Ms1 homolog in S. coelicolor ( 37 ) (Figure 6 A and B) and RIP-seq also verified the first nucleotide of scr3559 ( Figure 6 E and F) which we had determined by 5´RACE previously ( 37 ). ...
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 σfactors, 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.
... Recently, putative Ms1 RNAs homologs have been found among many actinobacteria, for example in Streptomyces coelicolor ( 36 ,37 ). In some actinobacteria, such as corynebacteria, neither 6S RNA nor Ms1 have been discovered so far ( 37 ) raising the question whether any similar RNA exists in these species. It is also unknown if both Ms1 and 6S RNA are present in Streptomyces coelicolor. ...
... 5´biotinylated oligonucleotide probes ( Supplementary Data ) were hybridized to the membrane and detected with the BrightStar BioDetect Kit (Ambion) or Novex or Tropix CDP STAR substrate (Ther-moFischer Scientific, Applied Biosystems) according to the manufacturer's instructions. 5´RACE and 3´RACE were performed according to the protocol used previously ( 37 ). ...
... Therefore, we also focused on RNAs associated with the transcriptional machinery in S. coelicolor A3(2) hrdB-HA, a strain in which the primary σ factor (HrdB) is endogenously tagged with hemagglutinin (HA) ( 38 ). We had previously shown that the anti-HA antibody immunoprecipitated the primary σ factor holoenzyme (HrdB-HA in complex with RNAP) ( 37 ). Here, we performed RNAP and HrdB-HA RIP-seq from exponential and stationary phases of growth (42 h and 66 h after germination, respectively, We confirmed that scr3559 sRNA is an Ms1 homolog in S. coelicolor ( 37 ) (Figure 6 A and B) and RIP-seq also verified the first nucleotide of scr3559 ( Figure 6 E and F) which we had determined by 5´RACE previously ( 37 ). ...
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.
... The latter phenotype is also consistent with another proposed function for HelD -storing inactive RNAPs in stationary phase (Pei et al., 2020). The sequestered RNAPs are reactivated when the cells encounter more advantageous conditions, reminiscent of similar roles of 6S and Ms1 sRNAs that sequester the primary factorcontaining RNAP holoenzyme or the RNAP core (subunit composition α 2 '), respectively (Wassarman, 2018, Vankova Hausnerova et al., 2022. ...
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.
