Structural Studies of Ribosome Regulation

核糖体调控的结构研究

• 批准号: 9505913
• 负责人: Christine M Dunham
• 金额: $ 34.01万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9505913
• 关键词: Address; Affect; Affinity; Amino Acids; Anticodon; Architecture; Bacteria; Binding Sites; Biochemical; Cell physiology; Cells; Codon Nucleotides; Collaborations; Complement; Complex; Cryoelectron Microscopy; DNA; Detection; Disease; Enzymes; Event; Funding; Gene Expression; Genetic Code; Genetic Translation; Genomics; Goals; Hand; Health; Human; Hydrolysis; Laboratories; Lead; Ligands; Link; Maintenance; Mediating; Mediator of activation protein; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Movement; Mutation; Nucleotides; Organism; Peptide Elongation Factor G; Peptides; Process; Production; Protein Biosynthesis; Proteins; Quality Control; RNA; Reading; Reading Frames; Regulation; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Sense Codon; Signal Transduction; Site; Structure; Suppressor Transfer RNA; Testing; Transfer RNA; Translational Regulation; Translations; Validation; X-Ray Crystallography; base; dalton; experimental study; genetic information; human disease; insight; interest; man; novel; particle; premature; prevent; public health relevance; recruit; release factor; stem; structural biology; translation factor

 DESCRIPTION (provided by applicant): Ribosomes are the complex, cellular machinery responsible for the production of all proteins in every living organism. This 2.5 million Dalton enzyme contains three large RNAs and more than 50 proteins that form two asymmetric subunits and promote mRNA-directed translation of the genetic code. Accurate translation requires the precise synchronization of regulatory factors, messenger RNAs and transfer RNAs to produce a mature protein. Errors associated with translation are detrimental to gene expression and hence cellular function. Furthermore, consistent with the critical importance of error- free protein synthesis for proper cellular function, there are numerous examples where human disease is linked to alterations in this macromolecular machinery that monitors the accuracy of these events. The major question that underlies translational regulation is how the ribosome is able to distinguish errors from non-canonical three-base decoding and tRNA misreading from normal function. Our long-term goal is to understand how this large macromolecular machine on a molecular level identifies such errors and how this process impacts human disease. This long-term goal will be addressed here by testing the hypothesis that mRNA and tRNA interactions with the ribosome cause conformational changes that prevent errors either through suppression of the mRNA mutation or via a new and novel proofreading mechanism for quality control purposes. Three independent but complementary aims are proposed. Experiments in Aim 1 will test if the +1 shift into the new frame is promoted by interactions with elongation factor G in the A site or by ribosomal components that structurally obstruct the tRNA path between the A and P site. In Aim 2 we will structurally characterize how fs tRNASufA6 interacts with the ribosomal P site and biochemically test whether mutations of important tRNA nts affect fs tRNAs affinity for the A site and/or how it is recognized by EF-G and moved from the A to the P site. These experiments build upon our new model for +1 frameshifting we established in the prior funding period. In Aim 3 we will investigate a second, possibly linked phenomenon: how mismatched P-site mRNA-tRNA interactions on the ribosome arising from tRNA selection errors lead to premature termination of protein synthesis. These aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes using both X-ray crystallography and single particle cryo-electron microscopy (in collaboration with Dr. Skiniotis) approaches and complementary biochemical methods.

 描述（由申请人提供）：核糖体是负责在每个活生物体中产生所有蛋白质的复杂细胞机器。这种250万道尔顿的酶含有三种大RNA和50多种蛋白质，它们形成两个不对称亚基，并促进mRNA指导的遗传密码翻译。准确的翻译需要调节因子、信使RNA和转移RNA的精确同步以产生成熟蛋白。与翻译相关的错误对基因表达有害，因此对细胞功能有害。此外，与无错误的蛋白质合成对于适当的细胞功能的至关重要性相一致，有许多例子表明人类疾病与这种监测这些事件的准确性的大分子机制的改变有关。翻译调控的主要问题是核糖体如何区分非典型的三碱基解码错误和正常功能的tRNA误读。我们的长期目标是了解这个大分子机器在分子水平上如何识别这些错误，以及这个过程如何影响人类疾病。这一长期目标将在这里通过测试的假设，即mRNA和tRNA与核糖体的相互作用，导致构象变化，防止错误，通过抑制mRNA突变或通过一个新的和新颖的校对机制，用于质量控制的目的来解决。提出了三个独立但互补的目标。目标1中的实验将测试+1向新框架的转变是否是通过与A位点中的延伸因子G的相互作用或通过在结构上阻碍A和P位点之间的tRNA路径的核糖体成分来促进的。在目标2中，我们将从结构上表征fs tRNASufA 6如何与核糖体P位点相互作用，并从生物化学上测试重要tRNA nt的突变是否影响fs tRNA对A位点的亲和力和/或它如何被EF-G识别并从A位点移动到P位点。这些实验建立在我们在上一个资助期建立的+1移码新模型的基础上。在目标3中，我们将研究第二个可能相关的现象：tRNA选择错误引起的核糖体上的错配P位点mRNA-tRNA相互作用如何导致蛋白质合成的过早终止。这些目标将通过使用X射线晶体学和单粒子冷冻电子显微镜（与Skiniotis博士合作）方法和互补生物化学方法的大型功能性核糖体复合物的结构生物学相结合来实现。