Structural Studies of Ribosome Regulation

 描述(申请人提供)：核糖体是复杂的细胞机械，负责在每个活着的有机体中生产所有蛋白质。这种250万的道尔顿酶包含三个大的RNA和超过50个蛋白质，形成两个不对称的亚基，促进基因密码的信使翻译。准确的翻译需要调控因子、信使RNA和转移RNA的精确同步才能产生成熟的蛋白质。与翻译相关的错误对基因表达和细胞功能都是有害的。此外，与无错误的蛋白质合成对正常细胞功能的关键重要性一致，有许多例子表明人类疾病与监测这些事件准确性的这个大分子机制的变化有关。翻译调控的主要问题是核糖体如何区分错误和非规范的三碱基解码，以及tRNA误读和正常功能。我们的长期目标是了解这台分子水平上的大分子机器如何识别此类错误，以及这一过程如何影响人类疾病。这一长期目标将在这里通过测试这样一个假设来解决，即mRNA和tRNA与核糖体的相互作用导致构象变化，通过抑制mRNA突变或通过用于质量控制目的的新的和新颖的校对机制来防止错误。提出了三个独立但相辅相成的目标。Aim 1的实验将测试A位点与延伸因子G的相互作用是否促进了+1向新框架的转变，或者是核糖体成分在结构上阻碍了A和P位点之间的tRNA路径。在目标2中，我们将从结构上表征fs tRNASufA6如何与核糖体P位点相互作用，并生化测试重要的tRNA NTS的突变是否影响A位点的fs tRNAs亲和力和/或它是如何被EF-G识别并从A位转移到P位的。这些实验建立在我们在前一个资助期建立的+1帧转换新模型的基础上。在目标3中，我们将研究第二个可能相关的现象：由于tRNA选择错误而引起的核糖体上不匹配的P-位点mRNA-tRNA相互作用如何导致蛋白质合成提前终止。这些目标将通过使用X射线结晶学和单颗粒冷冻电子显微镜(与Skiniotis博士合作)方法和补充生化方法的大型功能性核糖体复合体的结构生物学相结合来实现。

 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.