Structural studies of ribosome regulation

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。提出了两个独立但互补的目标。目的1试图了解一类新的突变tRNA如何与核糖体相互作用，以改变三碱基遗传阅读框架并抑制错误。目的2是为了了解一种新的质量控制机制的结构基础，导致tRNA：mRNA错配错误。这些目标将通过结合大型功能性核糖体复合物的结构生物学、生物化学和生物物理学方法来实现。

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. Two independent but complementary aims are proposed. Aim 1 seeks to understand how a novel class of mutant tRNAs interact with the ribosome to alter the three-base genetic reading frame and suppress errors. Aim 2 is designed to understand the structural basis of a new quality control mechanism resulting from tRNA:mRNA mismatch errors. These aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes, biochemical and biophysical methods.