Abstract
Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σA) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σA. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σA. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.
Highlights
Mycobacteria are an important group of bacteria that include lethal human pathogens-Mycobacterium tuberculosis and Mycobacterium leprae-and nonpathogenic saprophytic species, such as Mycobacterium smegmatis
We wanted to explore the relative amounts of Ms1 in M. smegmatis and compare it to the amounts of known 6S RNAs in E. coli and B. subtilis (recently reviewed in [23,24]). 6S RNA in E. coli (184 nt) and the main 6S RNA in B. subtilis are highly expressed during stationary phase and both are clearly visible when total RNA is resolved on denaturing PAGEs [13,20]
We found that almost 40% of the Ms1 in stationary cells was bound to mycobacterial core RNA polymerase (Figure 4D, the input represents the total amount of Ms1 isolated from the cell lysate; note that the amount of detected Ms1 depends on the amount of the antibody used)
Summary
Mycobacteria are an important group of bacteria that include lethal human pathogens-Mycobacterium tuberculosis and Mycobacterium leprae-and nonpathogenic saprophytic species, such as Mycobacterium smegmatis. The RNAP core associates with different σ factors that recognize different promoter sequences, and switching between these σ factors regulates gene expression. Bacteria have one primary (housekeeping) σ factor responsible for the majority of gene expression. This primary σ factor is called σ 70 in E. coli or σ A in M. smegmatis and Bacillus subtilis [4]. When conditions become unfavorable and bacteria enter stationary phase, the transcription of σ 70/σ A-dependent genes is reduced and genes recognized by alternative stress σ factors are activated [5]
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