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Role of the nascent TnaC Peptide in the Inhibition of the Ribosome Function by tryptophan

新生 TnaC 肽在色氨酸抑制核糖体功能中的作用

基本信息

批准号：
1158271
负责人：
Luis Cruz-Vera
金额：
$ 40.4万
依托单位：
University of Alabama in Huntsville
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2015-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1158271&HistoricalAwards=false
关键词：
Role nascent TnaC Peptide Inhibition

项目摘要

Intellectual Merit: Ribosomes are the molecular machines that carry out template-directed protein synthesis in all cells. They translate the sequence of the mRNA template into the corresponding protein sequence, in a process that is subject to multiple levels of regulation, including the ability to respond to the concentrations of certain cellular metabolites. An important but still poorly understood regulatory mechanism involves the interaction of the new protein (the 'nascent peptide') with specific cellular metabolites and with the ribosome itself, while the new protein is still near the site of synthesis inside the ribosome. The goal of this project is to elucidate how nascent peptides transform the ribosome into a metabolite-responsive machine to control protein synthesis, using as a model the regulation of the expression of the tnaCAB cluster of genes (operon) by the amino acid L-tryptophan. Previous studies suggest that when the nascent TnaC peptide of the tnaCAB operon is present in the ribosome, L-tryptophan can also bind, causing translation of the TnaC protein to stop, while increasing the expression of the downstream tnaA and tnaB genes, which encode an enzyme in the tryptophan degradation pathway and a L-tryptophan transporter, respectively. The planned work aims to identify the mechanism(s) by which the nascent TnaC peptide promotes L-tryptophan binding to the ribosome and arrest of translation of TnaC protein. The formation of the L-tryptophan binding site will be addressed by designing a method to covalently crosslink L-tryptophan with ribosome components in the presence of nascent TnaC peptides. To complement the information obtained by cross-linking, structural probing of ribosomal RNA will be carried out to localize changes induced by L-tryptophan binding. Determination of the mechanism(s) of inhibition by L-tryptophan will be studied by determining the dependence of the binding affinities of antibiotics and translation factors to TnaC-ribosome complexes as a function of L-tryptophan concentration. Also, the function of the ribosomes containing the nascent TnaC peptide and L-tryptophan will be tested using antibiotics, translational factors and organic solvents that are known to activate the ribosome. These assays will help discriminate between inhibition of the binding of these molecules to the ribosome and inhibition of the actual chemical reactions performed in the ribosome active site. The planned experiments will reveal which components of the ribosome are involved in detecting cellular metabolites and which functions of the ribosome are regulated by the nascent peptides to control the quality of protein synthesis and gene expression. Broader impact: Each year, this project will provide research training opportunities for two undergraduates majoring in genetics and two advanced graduate students training in biochemistry. In addition, a summer research experience will be offered annually to undergraduate students from Oakwood and Alabama A&M Universities, two schools in the northern Alabama region that predominantly serve under-represented minority populations. The PI and participating graduate students will contribute to already established workshops in genetics and biochemistry for students from Columbia High School, a minority-serving high school in the UA-Huntsville`s neighborhood. Ten high-school students interested in biology careers will be selected to serve as student instructors and will receive training in relevant concepts and experimental techniques from the PI and his graduate students. These students will serve as peer instructors to help the PI conduct workshops to reach a larger group of 40+ high-school students, and in the process deepen their own learning. Also, participating graduate students will help the PI improve biochemistry laboratory courses offered at the undergraduate and graduate levels at UA-Huntsville, by video recording laboratory procedures and lectures, which will be made available through the UA-Huntsville web-page for use by students, as well as by Columbia High School teachers. The active engagement of the PI, as a mentor and role model to the growing population of Hispanic-American students in the Huntsville area, will contribute signifiicantly to science education in North Alabama.

智力优点：核糖体是在所有细胞中进行模板指导的蛋白质合成的分子机器。它们将 mRNA 模板的序列翻译成相应的蛋白质序列，这一过程受到多个级别的调节，包括对某些细胞代谢物浓度做出反应的能力。一个重要但仍知之甚少的调节机制涉及新蛋白质（“新生肽”）与特定细胞代谢物和核糖体本身的相互作用，而新蛋白质仍然靠近核糖体内的合成位点。该项目的目标是阐明新生肽如何将核糖体转化为代谢物响应机器来控制蛋白质合成，以氨基酸 L-色氨酸对 tnaCAB 基因簇（操纵子）表达的调节为模型。先前的研究表明，当 tnaCAB 操纵子的新生 TnaC 肽存在于核糖体中时，L-色氨酸也可以结合，导致 TnaC 蛋白的翻译停止，同时增加下游 tnaA 和 tnaB 基因的表达，这两个基因分别编码色氨酸降解途径中的酶和 L-色氨酸转运蛋白。计划的工作旨在确定新生 TnaC 肽促进 L-色氨酸与核糖体结合并阻止 TnaC 蛋白翻译的机制。 L-色氨酸结合位点的形成将通过设计一种在新生 TnaC 肽存在的情况下将 L-色氨酸与核糖体成分共价交联的方法来解决。为了补充通过交联获得的信息，将对核糖体 RNA 进行结构探测，以定位 L-色氨酸结合引起的变化。将通过确定抗生素和翻译因子与 TnaC-核糖体复合物的结合亲和力与 L-色氨酸浓度的函数的依赖性来研究 L-色氨酸抑制机制的确定。此外，将使用已知可激活核糖体的抗生素、翻译因子和有机溶剂来测试含有新生 TnaC 肽和 L-色氨酸的核糖体的功能。这些测定将有助于区分对这些分子与核糖体结合的抑制和对核糖体活性位点中进行的实际化学反应的抑制。计划中的实验将揭示核糖体的哪些成分参与检测细胞代谢物，以及核糖体的哪些功能受到新生肽的调节，以控制蛋白质合成和基因表达的质量。更广泛的影响：每年，该项目将为两名遗传学专业的本科生和两名生物化学专业的高级研究生提供研究培训机会。此外，每年还将为奥克伍德大学和阿拉巴马农工大学的本科生提供暑期研究体验，这两所学校位于阿拉巴马州北部地区，主要服务于代表性不足的少数族裔群体。 PI 和参与的研究生将为哥伦比亚高中 (UA-Huntsville 附近的一所少数族裔高中) 的学生设立的遗传学和生物化学研讨会做出贡献。十名对生物学职业感兴趣的高中生将被选为学生导师，并接受 PI 及其研究生的相关概念和实验技术培训。这些学生将作为同伴导师，帮助 PI 举办研讨会，以覆盖更大范围的 40 多名高中生，并在此过程中加深自己的学习。此外，参与的研究生将通过录制实验室程序和讲座的视频，帮助 PI 改进 UA-Huntsville 本科生和研究生级别提供的生物化学实验室课程，这些课程将通过 UA-Huntsville 网页提供，供学生和哥伦比亚高中教师使用。作为亨茨维尔地区不断增长的西班牙裔美国学生的导师和榜样，PI 的积极参与将为北阿拉巴马州的科学教育做出重大贡献。