Regulation of the tryptophan genes in Bacillus

芽孢杆菌色氨酸基因的调控

基本信息

批准号：
6868781
负责人：
PAUL D GOLLNICK
金额：
$ 28.27万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-15 至 2008-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6868781
关键词：
Bacillus subtilis DNA footprinting RNA binding protein bacterial RNA bacterial genetics fluorescence spectrometry gene expression genetic regulation genetic transcription heat shock proteins protein binding protein protein interaction protein structure function site directed mutagenesis stop flow technique thermodynamics transcription factor tryptophan

项目摘要

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.

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细菌。这种控制机制统称为衰减和抗终止。拟议的研究将调查 RNA 结合蛋白识别和结合 RNA 中特定位点的机制，以及这些相互作用如何调节转录。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也将得到研究。研究的模型系统是 TRAP 蛋白，这是一种 RNA 结合蛋白，可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。当存在过量色氨酸时，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。 TRAP 是一种独特的已表征的 RNA 结合蛋白，因为它由排列成环状结构的 11 个相同亚基组成，并且它结合的 RNA 包含多达 11 个小（三核苷酸）重复元件。最近的研究表明 TRAP 通过两步机制与 RNA 结合。要测试的假设是 TRAP 首先与 RNA 的 5' 端结合，然后进行扫描，直到遇到结合位点的最多 5 个重复，此时形成起始复合物。随后将剩余的重复序列包裹在蛋白质环的外周上。 TRAP 与其 RNA 靶标结合的详细机制将通过结合动力学结合研究以及快速淬灭核酸酶保护和荧光研究来表征。一种称为抗 TRAP (AT) 的蛋白质特异性结合色氨酸激活的 TRAP，并抑制其与 RNA 的结合。将进行研究来表征 AT 的结构和功能，特别是它识别和结合 TRAP 的机制。