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.

dRNA polymerase (RNAP) is an extensively studied multisubunit enzyme required for transcription of DNA into RNA, yet the ␦ subunit of RNAP remains an enigmatic protein whose physiological roles have not been fully elucidated. Here, we identify a novel, so far unrecognized function of ␦ from Bacillus subtilis. We demonstrate that ␦ affects the regulation of RNAP by the concentration of the initiating nucleoside triphosphate ([iNTP]), an important mechanism crucial for rapid changes in gene expression in response to environmental changes. Consequently, we demonstrate that ␦ is essential for cell survival when facing a competing strain in a changing environment. Hence, although ␦ is not essential per se, it is vital for the cell's ability to rapidly adapt and survive in nature. Finally, we show that two other proteins, GreA and YdeB, previously implicated to affect regulation of RNAP by [iNTP] in other organisms, do not have this function in B. subtilis. RNA polymerase (RNAP) is the key enzyme responsible for transcription of DNA into RNA. Bacterial RNAP core enzyme consists of several subunits: the ␣ dimer that holds together ␤ and ␤=, which form the catalytic center, and the subunit that binds to ␤=. This core enzyme, ␣ 2 ␤␤=, is capable of elongating but not initiating transcription. To initiate transcription, the core enzyme must associate with a subunit that allows the holoenzyme to recognize specific sequences in the DNA, i.e., promoters. Typically, several different subunits are present in the cell and direct the expression of different subsets of genes (1, 2).While the ␣ 2 ␤␤= composition is conserved across the bacterial kingdom, Gram-positive Firmicutes contain an additional subunit, ␦, which is encoded by the rpoE gene in the model bacterium Bacillus subtilis. The ␦ subunit was first reported as an endogenous protein present in RNAP from phage SP01-infected Bacillus subtilis cells, which was required for its accurate middle gene transcription (3, 4). The rpoE gene specifies a protein of 173 amino acids (aa) with a molecular mass of ϳ20.5 kDa. The protein is highly acidic (pI, 3.6) (5). As determined by circular dichroism (CD) spectroscopy, it consists of two domains: (i) the N-terminal domain (NTD), which is structured; and (ii) the C-terminal domain, which is unstructured and whose amino acid composition-stretches of glutamic and aspartic acid residues-makes it virtually a polyanion (6). The structure of the ordered N-terminal domain was recently solved based on a truncated construct consisting of the N-terminal domain containing a His tag. The NTD contains four ␣-helices and an antiparallel ␤-sheet (7). Delta binds to RNAP in vivo (8), but the binding site is unknown.The in vitro effects of ␦ on transcription were previously examined in detail in the B. subtilis system. ␦ was reported to destabilize complexes between RNAP and DNA in vitro, thus increasing RNAP's specificity for good consensus promoter sequences (9, 10). Despite this inhibitory effect, ␦ was shown to stimulate transcription on some temp...