Molecular mechanisms of ribosome pausing and the cellular response

蛋白质合成在所有生命王国中是一个高度保守的过程，它可以被分解为四个 不同的阶段：起始、延伸、终止和核糖体再循环。这个广泛的目标 研究计划是使用生物化学和基因组方法来阐明共同的， 细菌中翻译延伸、终止和再循环的独特分子特征， 真核生物及其控制。在这里，我们特别关注翻译控制的一个方面， 该核糖体停滞触发细胞反应，导致mRNA衰变，靶向蛋白水解， 核糖体再循环特别是，我们最初集中在一个高度保守的失速基序，多碱基 肽序列，这是特别相关的真核细胞，其中替代聚腺苷酸化位点 使用通常导致"不停止"的mRNA。我们将继续使用体外生物化学和体内 核糖体分析，以了解这一生物学重要和保守过程的分子机制 (and其他相关系统）。更具体地说，我们建议（1）使用我们先前建立的体外 重组的翻译系统（与S.酿酒成分）提出一系列问题， 核糖体为基础的机制，用于传感翻译扰动，（2）使用一系列的报告， 酵母筛选有助于这些mRNA监视途径的新组分和（3）使用 核糖体分析方法用于定义生物学相关的体内靶，它们的分子特征， 以及促成这些重要途径的因素。我们预计， 方法将是强大的定义生物学相关的机制。

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.