Regulation of the tryptophan genes in Bacillus

描述(由申请人提供)：控制编码区上游的转录终止是调节细菌基因表达的常见策略，包括许多对人类健康重要的基因表达。这种控制机制统称为衰减和抗终止。这项拟议的研究将探讨RNA结合蛋白识别和结合RNA中特定位置的机制，以及这些相互作用如何调节转录。还将研究蛋白质-蛋白质相互作用调节RNA结合基因调节蛋白活性的机制。研究的模型系统是TRAP蛋白，这是一种RNA结合蛋白，调节枯草芽孢杆菌和相关细菌中色氨酸(Trp)基因的转录衰减。在过量色氨酸存在的情况下，TRAP被激活，与Trp操纵子mRNA的5‘前导区结合，并诱导转录终止子的形成，从而停止基因的表达。在已鉴定的RNA结合蛋白中，TRAP是独一无二的，因为它由11个相同的亚基组成环状结构，并且它结合包含多达11个小(三核苷酸)重复元件的RNA。最近的研究表明，TRAP通过两步机制与RNA结合。需要检验的假设是，TRAP首先与RNA的5‘端结合，然后扫描，直到它遇到结合位点的5个最多的重复，在这一点上形成了一个起始复合体。然后，将剩余的重复序列包裹在蛋白质环的外周。TRAP与其RNA靶标结合的详细机制将使用动力学结合研究以及快速猝灭核酸酶保护和荧光研究相结合的方法来表征。一种名为反TRAP(AT)的蛋白质特异性地与色氨酸激活的TRAP结合，并抑制它与RNA的结合。将对AT的结构和功能进行研究，特别是它识别和结合TRAP的机制。

DESCRIPTION (provided by applicant): Controlling transcription termination upstream of a coding region is a common strategy to regulate gene expression in bacteria, including many with importance to human health. Such control mechanisms are collectively termed attenuation and antitermination. The proposed research will investigate the mechanisms by which RNA binding proteins recognize and bind to specific sites in RNA, and how these interactions regulate transcription. The mechanism by which protein-protein interactions can modulate the activity of an RNA-binding gene regulatory protein will also be studied. The model system of study is the TRAP protein, an RNA binding protein that regulates transcription attenuation of the tryptophan (trp) genes in Bacillus subtilis and related bacteria. In the presence of excess tryptophan, TRAP is activated to bind to the 5' leader region of the trp operon mRNA and induce formation of a transcription terminator, which halts expression of the genes. TRAP is a unique among characterized RNA binding proteins in that it consists of 11 identical subunits arranged in a ring structure, and in that it binds RNAs that contain up to 11 small (trinucleotide) repeated elements. Recent studies indicate that TRAP binds to RNA by a two-step mechanism. The hypothesis to be tested is that TRAP first binds to the 5'-end of the RNA and then scans until it encounters the 5-most repeats of the binding site, at which point an initiation complex is formed. This is followed by wrapping the remainder of the repeats around the outer perimeter of the protein ring. The detailed mechanism by which TRAP associates with its RNA target will be characterized using a combination of kinetic binding studies, as well as rapid-quench nuclease protection and fluorescence studies. A protein called anti-TRAP (AT) specifically binds to tryptophan-activated TRAP and inhibits it from binding to RNA. Studies will be performed to characterize the structure and function of AT, particularly the mechanism by which it recognizes and binds to TRAP.