Elucidation of novel regulatory mechanisms employed by small noncoding RNAs from B. subtilis and identification of RNA chaperones involved in these mechanisms

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Whereas in Gram negative bacteria, systematic searches for small noncoding RNAs led to the discovery of a large number of such RNAs, much less is known from Gram positive bacteria. Using a computational approach, we could predict a number of potential small noncoding RNAs within the intergenic regions of the B. subtilis genome. Until now, two of these RNAs- SR1 and SR2 (renamed BsrF) - could be verified in subsequent Northern blotting analyses. SR1 and its interaction with the first identified primary target, ahrC mRNA, as well as further SR1 targets and CcpN, the transcriptional regulator of SR1, are investigated in project BR 1552/6-1 and 6-2 and 6/3. Meanwhile, we characterized the expression profile of SR2 (now BsrF) in detail and detected that the transcription of this RNA is activated by CodY about 3-fold in the presence of BCAA (branched chain amino acids) and GTP and slightly activated by glucose, but not by other sugars. Unfortunately, 4 independent Microarray analyses (3 in complex, 1 in CSE minimal medium) using BsrF wild-type and knockout vs. knockout and overexpression strain performed in Ulrike Mäders lab in Greifswald yielded completey different targets, all of which proved to be the wrong ones in our Norternblot or reporter gene analyses. These experiments bound a lot of time a material.. Hfq will no longer be the focus of the project since both SR1 and BsrF bound Hfq only at nonphysiologically high concentrations and their stability was not affected by Hfq at all. Furthermore, the result with the Hfq independent chaperone that bound BsrF could not be reproduced after moving to another lab and cultivating B. subtilis in our self-prepared culture media. Instead, in this project, we aim at: a) the identification of target(s) of BsrF and BsrG using three independently in parallel grown CSE minimal medium cultures containing BCAA (highest expression of BsrF) for 3 parallel microarray analyses and a subsequent statistical analysis as well as 454 sequencing with the same RNAs. In parallel, 2D-Gel electrophoreses should be performed that allow a comparision with the microarray data. The identification of the biological functions of BsrF and BsrG will be performed. b) the identification of further RNAs from our candidate list using a broad variety of growth and stress conditions, the characterization of such RNAs. c) the elucidation of novel mechanisms of action of small RNAs. The focus will be on sRNAs that - similar to SR1/ahrC – are not complementary to the SD sequences of their targets and, therefore, do not act by inhibition of translation initiation, but show complementarity in the central and 3’ part of the target or in a 5’ leader region far upstream from the ribosome binding site (possible attenuation mechanism).