Molecular mechanisms of ribosome pausing and the cellular response

描述(由申请人提供)： 项目摘要：蛋白质合成在所有生命王国中都是一个高度保守的过程，可分为四个不同的阶段：起始、延伸、终止和核糖体循环。这项研究计划的广泛目标是利用生化和基因组学方法阐明细菌和真核生物中翻译延长、终止和循环的共同和独特的分子特征及其控制。在这里，我们特别关注翻译控制的一个方面，在这个方面，核糖体停滞触发细胞反应，导致mRNA衰退、靶向蛋白分解和核糖体循环。特别是，我们关注一个高度保守的停滞基序，即在真核细胞中特别相关的多碱性多肽序列，在真核细胞中，替代的多聚腺苷基化位点的使用通常会导致“不间断”的mRNAs。我们将使用体外生物化学和体内核糖体图谱来研究这一具有生物重要性和保守性的过程的分子力学。更具体地说，我们建议(1)使用记者和我们之前建立的体外重组翻译系统(含有大肠杆菌和酿酒酵母成分)来询问一系列关于多碱基序列如何在伸长过程中扰乱核糖体功能的问题，(2)使用这些相同的记者和体外生物化学来定义不同的核糖体外因子如何与核糖体接触并影响延伸、终止和再循环，(3)使用最近发展的核糖体图谱方法来定义体内生物相关的靶标、它们的分子特征，以及在细胞中做出反应以解决危机的因素。我们预计，这些方法的协同作用将在定义生物相关机制方面发挥强大作用。

DESCRIPTION (provided by applicant): Project Summary: Protein synthesis is a highly conserved process in all kingdoms of life that can be broken down into four distinct phases: initiation, elongation, termination and ribosome recycling. The broad goals of this research program are to use biochemical and genomic approaches to shed light on the common and distinctive molecular features of translation elongation, termination, and recycling in bacteria and eukaryotes, and their control. Here we are particularly focused on one aspect of translational control in which ribosomal stalling triggers a cellular response leading to mRNA decay, targeted proteolysis, and ribosome recycling. In particular, we focus on a highly conserved stalling motif, the poly-basic peptide sequence that is of particular relevance in eukaryotic cells where alternative polyadenylation site usage commonly leads to "non-stop" mRNAs. We will use in vitro biochemistry and in vivo ribosome profiling to look at the molecular mechanics of this biologically important and conserved process. More specifically, we propose (1) to use reporters and our previously established in vitro reconstituted translation systems (with E. coli and S. cerevisiae components) to ask a series of questions about how poly-basic sequences disrupt ribosome function during elongation, (2) to use these same reporters and in vitro biochemistry to define how different extra-ribosomal factors engage the ribosome and impact elongation, termination and recycling and (3) to use recently developed ribosome profiling approaches to define the biologically relevant in vivo targets, their molecular features, and the factors that respond in the cell to resolve the crisis. We anticipate that the synergy of these approaches will be powerful in defining biologically relevant mechanism.