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

然而，在革兰氏阴性细菌中，系统地搜索小的非编码RNA导致发现了大量这样的RNA，而在革兰氏阳性细菌中所知的要少得多。使用一种计算方法，我们可以预测在枯草杆菌基因组的基因间隔区中有一些潜在的非编码小RNA。到目前为止，其中两个RNA-SR1和SR2(更名为BsrF)-可以在随后的Northern blotting分析中得到验证。在BR 1552/6-1、6-2和6/3项目中研究了SR1及其与第一个确定的主要靶标AHRC mRNA以及其他SR1靶标和转录调控因子CcpN的相互作用。同时，我们详细描述了SR2(现在的BsrF)的表达谱，发现在支链氨基酸(BCAA)和GTP存在的情况下，该RNA的转录被Cody激活约3倍，并被葡萄糖激活，但不被其他糖激活。不幸的是，在Greifswald的Ulrike Mäders实验室使用BsrF野生型和敲除菌株与敲除和过表达菌株进行的4个独立的微阵列分析(3个在复合体中，1个在CSE最小培养基中)产生了完全不同的目标，所有这些都被证明是我们的Nortern印迹或报告基因分析中错误的。这些实验把一种材料捆绑了很长时间。Hfq将不再是该项目的重点，因为SR1和BsrF仅在非生理性的高浓度下与Hfq结合，其稳定性根本不受Hfq的影响。此外，转移到另一个实验室并在自制的培养基上培养枯草杆菌后，与BsrF结合的Hfq独立伴侣的结果不能重现。相反，在这个项目中，我们的目标是：a)使用三种平行生长的含有BsrF(BsrF的最高表达)的CSE最小培养基中的三种独立培养物来鉴定BsrF和BsrG的靶标(S)，用于3个平行的微阵列分析和随后的统计分析以及454个相同RNA的测序。同时，应进行2D-Gel电泳法，以便与微阵列数据进行比较。将对BsrF和BSRG的生物学功能进行鉴定。B)使用各种各样的生长和胁迫条件从我们的候选名单中鉴定出更多的RNA，这些RNA的特征。C)阐明小RNA的新作用机制。重点将放在与SR1/AHRC相似的sRNA上，这些sRNA与其靶标的SD序列不互补，因此不通过抑制翻译起始起作用，但在靶标的中心和3‘部分或在远离核糖体结合位点上游的5’前导区显示互补(可能的衰减机制)。

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).