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控，包括对某些细胞代谢物浓度作出反应的能力。一个重要的但仍然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以控制蛋白质合成，使用氨基酸L-色氨酸对tnaCAB基因簇（操纵子）表达的调节作为模型。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，它们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在新生TnaC肽存在下使L-色氨酸与核糖体组分共价交联的方法来解决。为了补充通过交联获得的信息，将进行核糖体RNA的结构探测以定位由L-色氨酸结合诱导的变化。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-色氨酸浓度的关系，研究L-色氨酸抑制机制的确定。此外，含有新生TnaC肽和L-色氨酸的核糖体的功能将使用已知激活核糖体的抗生素、翻译因子和有机溶剂进行测试。这些测定将有助于区分这些分子与核糖体结合的抑制和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组分参与检测细胞代谢物，以及核糖体的哪些功能受新生肽的调节，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，每年还将向奥克伍德大学和亚拉巴马A M大学的本科生提供夏季研究经验，这两所大学位于亚拉巴马北方地区，主要为代表性不足的少数民族人口提供服务。PI和参与的研究生将为来自哥伦比亚高中的学生在遗传学和生物化学方面已经建立的研讨会做出贡献，哥伦比亚高中是一所位于UA-Huntsville附近的少数民族高中。十名对生物学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。这些学生将担任同侪导师，以帮助PI进行研讨会，以达到更大的群体40+高中学生，并在此过程中加深自己的学习。此外，参与研究生将帮助PI改善在本科和研究生水平在UA-亨茨维尔提供的生物化学实验室课程，通过视频记录实验室程序和讲座，这将通过UA-亨茨维尔网页提供给学生使用，以及由哥伦比亚高中教师。PI的积极参与，作为一个导师和榜样，在亨茨维尔地区的拉美裔美国学生的人口不断增长，将有助于显着北亚拉巴马的科学教育。

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.