Molecular mechanisms of ribosome pausing and the cellular response

蛋白质合成在所有生命王国中都是一个高度保守的过程，可以分为四个阶段 不同的阶段：启动、延长、终止和核糖体循环。这个项目的总体目标是 研究计划是使用生化和基因组方法来阐明共同和 细菌和细菌中翻译延伸、终止和循环的独特分子特征 真核生物和它们的控制。在这里，我们特别关注翻译控制的一个方面 核糖体的停滞会触发细胞反应，导致信使核糖核酸的衰退、靶向蛋白降解，以及 核糖体回收。特别是，我们最初关注的是一个高度保守的拖延基序，即多碱基 多肽序列，这在真核细胞中具有特殊的相关性，其中可替代多聚腺苷基化位点 使用通常会导致“不间断”的mRNAs。我们将继续使用体外生物化学和体内 核糖体图谱以观察这一生物上重要和保守的过程的分子力学 (以及其他相关系统)。更具体地说，我们建议(1)使用先前在体外建立的 重组的翻译系统(包含酿酒酵母组件)，以提出一系列关于 基于核糖体的翻译扰动感知机制，(2)使用一系列记者在 酵母菌筛选有助于这些信使核糖核酸监测途径的新成分和(3)使用 核糖体图谱方法，以确定生物相关的体内靶点，它们的分子特征， 以及促成这些重要途径的因素。我们预计，这些因素的协同作用 在定义生物相关机制方面，方法将是强有力的。

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 focused initially 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 continue to use in vitro biochemistry and in vivo ribosome profiling to look at the molecular mechanics of this biologically important and conserved process (and other related systems). More specifically, we propose (1) to use our previously established in vitro reconstituted translation system (with S. cerevisiae components) to ask a series of questions about ribosome-based mechanisms for sensing translational perturbations, (2) to use a series of reporters in yeast to screen for novel components that contribute to these mRNA surveillance pathways and (3) to use ribosome profiling approaches to define the biologically relevant in vivo targets, their molecular features, and the factors that contribute to these important pathways. We anticipate that the synergy of these approaches will be powerful in defining biologically relevant mechanism.