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

阐明来自枯草芽孢杆菌的小非编码 RNA 采用的新调控机制，并鉴定参与这些机制的 RNA 伴侣

• 批准号: 40035314
• 负责人: Privatdozentin Dr. Sabine Brantl
• 金额: --
• 依托单位: Lehrstuhl für Genetik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/40035314?language=en
• 关键词: Elucidation; novel; regulatory; mechanisms; employed

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).

而在革兰氏阴性细菌中，系统搜索小的非编码RNA导致发现了大量的此类RNA，而革兰氏阳性细菌中所知的要少得多。使用计算方法，我们可以预测B基因间区域内的一些潜在的小非编码RNA。枯草杆菌基因组到目前为止，这些RNA中的两种-SR 1和SR 2（重命名为BsrF）-可以在随后的北方印迹分析中得到验证。在项目BR 1552/6-1和6-2和6/3中研究了SR 1及其与第一个鉴定的主要靶标ahrC mRNA以及其他SR 1靶标和SR 1的转录调节因子CcpN的相互作用。同时，我们详细表征了SR 2（现在的BsrF）的表达谱，并检测到该RNA的转录在BCAA（支链氨基酸）和GTP存在下被CodY激活约3倍，并被葡萄糖轻微激活，但不被其他糖激活。不幸的是，在Greifswald的Ulrike Mäders实验室中使用BsrF野生型和敲除与敲除和过表达菌株进行的4次独立的微阵列分析（3次在复合物中，1次在CSE基本培养基中）产生了完全不同的靶标，所有这些都被证明是我们Norternblot或报告基因分析中的错误靶标。这些实验花费了大量的时间来研究一种材料。Hfq将不再是该项目的重点，因为SR 1和BsrF仅在非生理高浓度下结合Hfq，并且它们的稳定性根本不受Hfq的影响。此外，在转移到另一个实验室并培养B后，不能再现具有结合BsrF的Hfq非依赖性伴侣的结果。枯草芽孢杆菌在我们自己准备的培养基。相反，在这个项目中，我们的目标是：a）使用三个独立平行生长的含有BCAA（BsrF的最高表达）的CSE基本培养基培养物鉴定BsrF和BsrG的靶标，用于3个平行的微阵列分析和随后的统计分析以及用相同RNA的454个测序。与此同时，应该进行2D凝胶电泳，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）使用多种生长和应激条件从我们的候选物列表中鉴定另外的RNA，表征这些RNA。c）阐明小RNA的新作用机制。重点将放在sRNA上，类似于SR 1/ahrC，它们与其靶标的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'前导区中显示互补性（可能的减毒机制）。