MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

TRAP-RNA识别调控TRP基因的机制

• 批准号: 6019063
• 负责人: PAUL L BABITZKE
• 金额: $ 16.67万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019063
• 关键词: Bacillus subtilis; RNA binding protein; SDS polyacrylamide gel electrophoresis; bacterial RNA; bacterial genetics; bacterial proteins; double stranded RNA; gel mobility shift assay; gene mutation; genetic regulation; genetic translation; nucleic acid repetitive sequence; nucleotides; operon; protein structure function; ribosomes; transcription termination

Numerous studies have provided a detailed understanding of how regulatory proteins interact with DNA, however relatively little is known about the principles that dictate how sequence-specific RNA-binding proteins recognize their RNA targets. Since fragile X syndrome is caused by defects in an RNA-binding protein, and RNA-binding proteins are involved in regulating HIV and adenovirus gene expression, results from the proposed studies could have a Positive impact on human health. TRAP of Bacillus subtilis is responsible for regulating expression of the trpEDCFBA operon and trpG by binding to a series of closely spaced G/UAG repeats present in each transcript. TRAP binds to a segment of the trp leader RNA that includes the antiterminator, thereby promoting transcription termination. TRAP also binds to a segment of the trpG transcript that includes the trpG ribosome binding site (RBS). The mechanisms responsible for these regulatory events will be characterized using a combination of genetic and biochemical approaches. The first aim of the proposed research is to determine the nucleotides in the G/UAG repeat sequences that are required for TRAP binding, to define the nucleotide spacing requirements between adjacent repeats, and to determine the number of repeats that are necessary to form a stable TRAP-RNA complex. This will be accomplished by testing the ability of various artificial RNA targets to serve as TRAP binding sites in a filter binding assay. Similar experiments will be performed to determine if TRAP can bind to double-stranded RNA in addition to single-stranded RNA. The next aim is to demonstrate that TRAP binding to the trpG RBS prevents translation. RNA footprint analyses will be performed to identify the nucleotides of the trpG transcript that TRAP and ribosomes interact with, and to demonstrate that bound TRAP prevents ribosome binding. In vitro translation studies will be carried out to determine if TRAP binding inhibits TrpG synthesis. The next goal is to determine the time frame in which TRAP binds to the nascent trp leader transcript. If TRAP binding occurs after the antiterminator is formed it is likely that RNA polymerase pauses following its formation to prevent readthrough past the termination signal. In the absence of pausing, TRAP binding must be sufficiently rapid to prevent antiterminator formation. Single-round transcription experiments will be carried out in vitro to determine if RNA polymerase pauses following antiterminator formation. NusA protein will be added to the transcription system to see if it plays a role in polymerase pausing. Evidence for pausing will also be examined in vivo. Lastly, a structure-function analysis will be performed to identify amino acid residues of TRAP that are responsible for RNA binding, L-tryptophan binding, and subunit oligomerization, as well as to identify nucleotides that interact with specific amino acid residues of TRAP. TRAP deficient mutants will be obtained using several strategies, including genetic selections and site-directed methods. The defects associated with various mutant TRAP proteins will be determined using available techniques. A collaboration with William Royer will continue to determine the crystal structure of the TRAP-RNA complex.

许多研究提供了一个详细的了解如何监管 蛋白质与DNA相互作用，但对蛋白质与DNA的相互作用知之甚少。 决定序列特异性RNA结合蛋白 识别它们的RNA靶标。由于脆性X综合征是由缺陷引起的 在RNA结合蛋白中，RNA结合蛋白参与 调节艾滋病毒和腺病毒基因表达，结果从建议 研究可能对人类健康产生积极影响。芽孢杆菌的TRAP 枯草杆菌负责调节trpEDCFBA操纵子的表达 和trpG通过结合一系列紧密间隔的G/UAG重复存在于 每一份成绩单TRAP与trp前导RNA的一段结合， 包括抗终止子，从而促进转录终止。 TRAP还结合到包含trpG的trpG转录物的片段 核糖体结合位点（RBS）。负责这些问题的机制 调控事件将使用遗传和 生物化学方法。拟议研究的第一个目标是 确定所需的G/UAG重复序列中的核苷酸 对于TRAP结合，以定义TRAP和TRAP之间的核苷酸间隔要求。 相邻的重复序列，并确定重复序列的数量， 形成稳定的TRAP-RNA复合物所必需的。这将通过 测试各种人工RNA靶标作为TRAP的能力 过滤器结合测定中的结合位点。类似的实验将在 以确定TRAP是否可以结合双链RNA， 单链RNA。下一个目标是证明TRAP结合 RBS阻止了翻译。RNA足迹分析将是 进行鉴定TRAP和TRAP的trpG转录物的核苷酸， 核糖体相互作用，并证明结合TRAP防止 核糖体结合将进行体外翻译研究， 确定TRAP结合是否抑制TrpG合成。下一个目标是 确定TRAP与新生trp前导序列结合的时间范围 成绩单。如果TRAP结合发生在抗终止子形成之后， RNA聚合酶可能在其形成后暂停， 通过终止信号进行通读。在没有停顿的情况下，TRAP 结合必须足够快以防止抗终止子形成。 将在体外进行单轮转录实验， 确定RNA聚合酶是否在抗终止子形成后暂停。 NusA蛋白将被添加到转录系统中， 在聚合酶暂停中的作用。暂停的证据也将被审查 in vivo.最后，将进行结构-功能分析， 鉴定负责RNA结合的TRAP的氨基酸残基， L-色氨酸结合和亚基寡聚化，以及鉴定 与TRAP的特定氨基酸残基相互作用的核苷酸。陷阱 将使用几种策略获得缺陷突变体，包括 遗传选择和定点方法。与以下方面相关的缺陷 各种突变的TRAP蛋白将使用可获得的 技术.与威廉·罗耶的合作将继续确定 TRAP-RNA复合物的晶体结构。