Dokument

Summary:
6S-1 RNA (bsrA) is a 190 nucleotides small non-coding RNA found in Bacillus subtilis, which binds to the housekeeping sigma A (σA) RNA polymerase holoenzyme (RNAP). 6S-1 RNA levels peak in stationary phase where the RNA supports adaptation to nutrient scarcity (or other stresses) and prepares cells for an instant outgrowth from stationary phase upon nutrient re-supply. In addition to inhibiting cellular transcription by binding to σA-RNAP, 6S-1 RNA also serves as a template for this enzyme to direct the synthesis of small product RNAs (pRNAs) in an RNA-dependent RNA polymerization reaction. These pRNAs, if long enough (~ 14-mer), are able to rearrange the structure of 6S-1 RNA such that the polymerase is released from the 6S-1 RNA and transcription of DNA promoters can be resumed. The kinetics of the process and key nucleotides involved in release and rearrangement of 6S-1 are unknown. Towards a deeper understanding of this process, we introduce substitutions/deletions along the secondary structure of 6S-1 RNA and analyse the impact thereof on the kinetics of 6S-1 rearrangement. In our study, we found that mutations C44/45 in the 5’-Central Bulge (CB) weaken 6S-1 RNA:σA-RNAP ground state binding two- to threefold while stabilizing the Central Bulge Collapse Helix (CBC) and shifting the pRNA length pattern to shorter pRNAs. A 6S-1 RNA variant with a weakened helix P2 and CBC stabilized by the the C44/45 mutation was more effectively rearranged and released from the enzyme. Our mutational analysis also revealed that formation of a second short hairpin in 3’-CB is detrimental to 6S-1 RNA release. From our results we infer that formation of the CBC subtly supports pRNA-induced 6S-1 RNA rearrangement and release. Additionally, truncated variants of 6S-1 RNA, solely consisting of the CB flanked by two short helical arms, can still traverse the functional 6S RNA cycle in vitro, despite decreased σA-RNAP affinity. This indicates that the ‘- 35-like’ region is not strictly essential for 6S-1 RNA function, at least in B. subtilis. We also showed that pRNA-isosequential locked nucleic acids (pLNAs) as short as 6 nt were able to induce 6S-1 RNA rearrangement and disrupt/prevent complex formation with σA-RNAP. Additionally, we analyzed the spatial dimensions of free 6S-1 RNA versus 6S-1:pLNA complexes by atomic force microscopy, revealing that 6S-1:pRNA hybrid structures, on average, adopt a more bent/constrained structure than 6S-1 RNA alone. The pLNA studies help us interpret the 6S-1 rearrangement to be a progressive process with intermediate states, ultimately leading to a maximally constrained and bent structure. Finally, we observed that the pRNA-mediated rearrangement of 6S-1 RNA and its release from σA-RNAP largely accelerates at NTP concentrations > 40 μM, which supports the role of 6S-1 RNA during nutrient re-supply. In gel assays, the regulatory 6S-1 and 6S-2 RNAs of Bacillus subtilis bind to the housekeeping RNA polymerase holoenzyme (σA-RNAP) with submicromolar affinity. We observed copurification of endogenous 6S RNAs from a published B. subtilis strain expressing a His-tagged RNAP. Such 6S RNA contaminations in σA-RNAP preparations reduce the fraction of enzymes that are accessible for binding to DNA promoters. In addition, this leads to background RNA synthesis by σA-RNAP utilizing copurified 6S RNA as template for the synthesis of short abortive transcripts termed product RNAs (pRNAs). To avoid this problem, we constructed a B. subtilis strain expressing His-tagged RNAP but carrying deletions of the two 6S RNA genes. The His-tagged, 6S RNA-free σA-RNAP holoenzyme can be prepared with sufficient purity and activity by a single affinity step. We also reported expression and separate purification of B. subtilis σA that can be added to the His-tagged RNAP to maximize the amount of holoenzyme and, by inference, in vitro transcription activity. In order to test our hypothesis that the regulatory role of 6S RNAs may be particularly important under natural, constantly changing environmental conditions, we constructed 6S RNA deletion mutants of the undomesticated B. subtilis wild-type strain NCIB 3610. We observed a strong phenotype under stress conditions in which the Δ6S-2 RNA strain exhibited retarded swarming activity and earlier spore formation. In contrast, the Δ6S-1&2 double knockout strain exhibited lesser spore formation than the wild-type. Additionally, we could show that 6S mutants grown under nutrient rich conditions revealed no strong phenotype. Our data suggests that both 6S RNAs contributes to the fitness of B. subtilis under the unsteady and temporarily harsh conditions encountered in natural habitats.

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