Regulation of the tryptophan genes in Bacillus

芽孢杆菌色氨酸基因的调控

• 批准号: 7149167
• 负责人: PAUL D GOLLNICK
• 金额: $ 26.9万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 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/7149167
• 关键词: Affect; Bacillus (bacterium); Bacillus subtilis; Bacteria; Binding; Binding Sites; Biochemical Genetics; Biological Models; Code; Collaborations; Complex; Elements; Fluorescence; Gene Expression; Genes; Genetic Transcription; Goals; Health; Human; Kinetics; Messenger RNA; Operon; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Regulation; Research; Resolution; Scanning; Site; Structure; Surface Plasmon Resonance; System; Testing; Thermodynamics; Transfer RNA; Transfer RNA Aminoacylation; Translations; Trinucleotide Repeats; Tryptophan; antitermination; attenuation; genetic regulatory protein; novel; nuclease; protein protein interaction; response; stopped-flow fluorescence; transcription termination

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基因。这种控制机制统称为衰减和反终止。提出的研究将探讨RNA结合蛋白识别和结合RNA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结合基因调控蛋白活性的机制也将被研究。研究的模型系统是TRAP蛋白，一种在枯草芽孢杆菌和相关细菌中调节色氨酸（trp）基因转录衰减的RNA结合蛋白。在色氨酸过量的情况下，TRAP被激活，与色氨酸操纵子mRNA的5'先导区结合，并诱导形成转录终止子，从而停止基因的表达。TRAP是一种独特的RNA结合蛋白，它由11个排列成环状结构的相同亚基组成，并且它结合含有多达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。要验证的假设是，TRAP首先与RNA的5'端结合，然后扫描，直到遇到结合位点的5次重复，此时形成起始复合物。接下来是将剩余的重复序列包裹在蛋白质环的外周长。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、快速猝灭核酸酶保护和荧光研究来表征。一种叫做anti-TRAP （AT）的蛋白质特异性地与色氨酸激活的TRAP结合，并抑制其与RNA的结合。将进行研究，以表征AT的结构和功能，特别是它识别和结合TRAP的机制。