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

智力优势：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，这一过程受到多个水平的调节，包括对某些细胞代谢物浓度做出反应的能力。一种重要但仍鲜为人知的调控机制涉及新蛋白质(“新生多肽”)与特定细胞代谢物和核糖体本身的相互作用，而新蛋白质仍在核糖体内合成部位附近。本项目的目标是阐明新生多肽如何将核糖体转化为代谢反应机器来控制蛋白质合成，并以氨基酸L-色氨酸对tna CAB基因簇(操纵子)表达的调控为模型。以前的研究表明，当核糖体中存在tNACAB操纵子的新生TNAC肽时，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一个酶和L-色氨酸转运体。计划中的工作旨在确定新生TNAC肽促进L-色氨酸与核糖体结合和阻止TNAC蛋白翻译的机制(S)。L-色氨酸结合位点的形成将通过设计一种方法来解决，该方法在新生TNAC多肽存在的情况下将L-色氨酸与核糖体成分共价交联。为了补充通过交联获得的信息，将进行核糖体RNA的结构探测，以定位L-色氨酸结合引起的变化。确定L-色氨酸的抑制机制(S)将通过测定抗生素和翻译因子与TNAC-核糖体复合体的结合亲和力随L-色氨酸浓度的变化而进行研究。此外，还将使用抗生素、翻译因子和已知激活核糖体的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。这些分析将有助于区分抑制这些分子与核糖体的结合和抑制核糖体活性部位进行的实际化学反应。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，核糖体的哪些功能受到新生多肽的调节，以控制蛋白质合成和基因表达的质量。更广泛的影响：该项目每年将为两名遗传学专业本科生和两名生物化学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。奥克伍德大学和阿拉巴马农工大学位于阿拉巴马州北部，主要服务于未被充分代表的少数族裔群体。PI和参与其中的研究生将为哥伦比亚高中的学生们已经建立的遗传学和生物化学研讨会做出贡献。哥伦比亚高中是一所为亚利桑那州亨茨维尔附近的少数民族服务的高中。10名对生物职业感兴趣的高中生将被选为学生导师，并将接受PI及其研究生的相关概念和实验技术培训。这些学生将担任同伴导师，帮助PI举办研讨会，接触到更大的40多名高中生，并在此过程中深化自己的学习。此外，参与的研究生将通过录像实验室程序和讲座来帮助PI改进在UA-Huntsville本科生和研究生水平提供的生物化学实验室课程，这些课程将通过UA-Huntsville网页提供给学生和哥伦比亚高中的教师使用。作为亨茨维尔地区不断增长的拉美裔美国学生的导师和榜样，PI的积极参与将对北阿拉巴马州的科学教育做出重大贡献。

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.