Characterization of trigger enzymes involved in the regulation and function of sRNAs in B. subtilis

到目前为止，人们只知道几种所谓的触发酶。这些是新陈代谢酶，完成控制基因表达的第二个功能。迄今已知的触发酶可以结合DNA并作为转录因子，通过结合RNA来影响RNA的稳定性，通过磷酸化来修饰蛋白质，或者通过蛋白质/蛋白质相互作用来抑制转录因子。此外，与大肠杆菌和其他革兰氏阴性细菌相比，人们对革兰氏阳性细菌，特别是枯草杆菌中的sRNA及其最新目标了解不多。在后一种细菌中，只有5个sRNA靶标被确定，其中包括我们组研究的两个sRNA SR1和SR4。在这个项目中，我们想要a)表征一种新的触发酶，丙酮酸羧基酶A(PycA)，它是我们课题组最近发现的sRNA SR1的第二调节因子，非常详细的生化和关于它的生物学作用。B)进一步确定PYCA的作用靶点，并分析触发酶在这些靶基因启动子上的作用。c)证明枯草杆菌甘油醛-3-磷酸脱氢酶GAPA也是一个触发酶(RNase或与RNase或降解体成分相互作用的蛋白质)，其功能受SR1d编码的小肽SR1P的调节。进一步鉴定其稳定性依赖于GAPA的RNA，并研究GAPA在这些RNAs中的作用机制。

So far, only a few so called trigger enzymes are known. These are metabolic enzymes that fulfill a second function in control of gene expression. Trigger enzymes known to date can e.g. bind DNA and act as transcription factors, affect the stability of RNA by binding it, modify proteins by e.g. phosphorylation or inhibit transcription factors by protein/protein interactions. Furthermore, compared to E. coli and other Gram-negative bacteria, not so much is known about sRNAs and their targest in Gram-positive bacteria, in particular B. subtilis. In the latter bacterium, only for five sRNAs targets have been identified, among them for the two sRNAs SR1 and SR4 investigated in our group.In this project we want toa) characterize a novel trigger enzyme, pyruvate carboxylase A (PycA) recently identified in our group as second regulator of the sRNA SR1, in great detail biochemically and concerning its biological role. Both the mechanism of action and the structural and biological prerequisites for its action should be elucidated.b) identify further targets of PycA and analyse the action of this trigger enzymes at the promoters of these target gens.c) demonstrate that glyceraldehyde-3-phosphate-dehydrogenase GapA from B. subtilis is also a trigger enzyme (either a RNase or a protein that interacts with an RNAse or components of the degradosome), whose function is modulated by the small peptide SR1P encoded by SR1d) identify further RNAs whose stability depends on GapA, and investigate the mechanism of action of GapA at these RNAs.