Ms1 RNA increases the amount of RNA polymerase in Mycobacterium smegmatis

Abstract

Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, P Ms1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β′ subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.

Regulation of ribosomal protein synthesis in mycobacteria: autogenous control ofrpsO

“…4B). The same was previously shown for the Msm rrnB promoter (28); in addition, the promoters of the sRNAs Ms1 (45) and MrsI (46) were also reported to be active in 5’ extended versions. The exact mechanism for the enhancement of transcription efficiency upon the promoter extending has not yet been clarified.…”

“…Like the rrnB (28) or Ms1 (45) core promoters, the rpsO core promoter of M. smegmatis appeared rather weak requiring the upstream sequences for stimulating its activity. As the mycobacterial rpsO genes are transcribed from a single promoter, according to the RNAseq data (47, 48), the stimulation by the upstream sequences cannot be due to the activity of additional upstream promoters but rather may be explained by the action of as-yet-unknown transcription factors the binding sites of which are situated within the upstream sequence (upstream activating elements).…”

“…3B). Analysis of the published data revealed that, in contrast to E. coli , mycobacterial core promoters (including only −10 and −35 promoter regions) may be inefficient requiring 5’extensons to augment their strength (28, 45, 46). In particular, the core Msm rrnB promoter (which a priori should be one of the strongest in bacterial cells) remained relatively weak unless the upstream region was significantly extended (28).…”

“…The exact mechanism for the enhancement of transcription efficiency upon the promoter extending has not yet been clarified. One of the reasonable explanations is the existence of the upstream binding sites for as-yet unknown transcription factors acting as activators (28, 45). Based on the experimental observations, we used the Msm rpsO - egfp fusion bearing the −231 extension for further experiments.…”

Resuscitation of Dormant “Non-culturable” Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst

“…MTS2823 belonging to 6S ncRNAs is the most abundant ncRNA in the stationary phase of M. tuberculosis (Arnvig et al, 2011). Its M. smegmatis homolog Ms1 influences the RNApolymerase level and is suggested to sequester the RNA polymerase core in a cache of inactive enzymes, which can be reactivated when needed (Sikova et al, 2019). Such a mechanism could be critical when the demand for RNA polymerase activity increases after environmental changes such as nutrient availability and outgrowth from the stationary phase.…”