Modulation of translation by synonomous codons in yeast

项目摘要 从mRNA到蛋白质的遗传密码的翻译最终决定了细胞的蛋白质组成。 牢房翻译延伸及其保真度对人类健康至关重要，因为翻译因子的突变 会导致智力残疾翻译延伸受同义密码子的选择调节 用于编码多肽，并经受多种质量控制机制以防止多肽的合成。 异常蛋白质在酿酒酵母中，CGA-CGA密码子对的翻译被强烈地抑制。 抑制性，比任何单一密码子都强。抑制由核糖体蛋白Asc 1（人）介导 RACK 1），当核糖体碰撞时，其触发核糖体质量控制（RQC）系统的接合。到 为了定义密码子介导的翻译效应的范围和机制，我们最近使用了一个高 GFP变体的通量测定，以鉴定17个强抑制性密码子对，其中12个是在 最慢翻译的密码子对。我们推断，这些对在功能上是重要的，因为大多数 慢翻译对在近缘物种中基因的相应位置高度保守。 我们还在研究阅读帧维护的关键过程，这是 核糖体我们发现缺乏Asc 1的菌株在CGA密码子重复序列上发生了广泛的移码。 利用遗传选择，我们最近发现了另外两种蛋白质，它们与Asc 1共同作用， CGA重复序列的移码：uS 3/Rps 3，一种普遍保守的核糖体蛋白，和Mbf 1，一种 古细菌/真核生物保守蛋白，其在翻译中的作用知之甚少。尽管深入研究 阅读帧维护，这整个系统涉及Asc 1、Mbf 1和Rps 3，具体到 真核生物从未被研究过 另外的初步结果表明，另外两种蛋白参与了阅读码框的维持：eS 26和 GCN 1。核糖体蛋白eS 26位于碰撞的核糖体的界面，最近被牵连 在框架转换中。gcn 1是一个保守的应激反应途径的主要调节因子，参与感知 当核糖体由于氨基酸饥饿而停滞时，核糖体A位点上的不带电荷的tRNA。 值得注意的是，12个最具抑制性的密码子对中有3个对RQC系统有反应，并且需要Mbf 1 对于阅读码框维持，而其它9个抑制性密码子对不具有这种功能。这些机制 九对对翻译发挥其影响是一个谜，但似乎可能涉及的核心组成部分， 翻译控制系统，因为这些对中的许多是高度保守的并且缓慢翻译。 为了跟进这些结果，我们建议1。确定Mbf 1、Rps 3和Asc 1 努力保持阅读框架。2.研究Rps 26和Gcn 1蛋白在移码中的作用。3. 定义不同的抑制密码子对发挥其作用的机制。

Project Summary Translation of the genetic code from mRNA into protein ultimately determines the protein composition of the cell. Translation elongation and its fidelity are essential for human health, as mutations in translation factors can result in intellectual disability. Translation elongation is modulated by the choice of synonymous codons used to encode a polypeptide and is subject to multiple quality control mechanisms to prevent synthesis of aberrant proteins. In the yeast Saccharomyces cerevisiae, translation of CGA-CGA codon pairs is strongly inhibitory, much more so than any single codon. Inhibition is mediated by ribosomal protein Asc1 (human RACK1), which triggers engagement of the ribosome quality control (RQC) system when ribosomes collide. To define the scope and mechanisms of codon-mediated effects on translation, we recently used a high throughput assay of GFP variants to identify 17 strongly inhibitory codon pairs, 12 of which are among the most slowly translated codon pairs in yeast. We infer that these pairs are functionally important, as the most slowly translated pairs are highly conserved in the corresponding positions of genes in closely related species. We are also studying the crucial process of reading frame maintenance, one of the most basic functions of the ribosome. We had found that strains lacking Asc1 undergo extensive frameshifting at CGA codon repeats. Using a genetic selection, we recently identified two additional proteins that work together with Asc1 to prevent frameshifting at CGA repeats: uS3/Rps3, a universally conserved ribosomal protein, and Mbf1, an archaeal/eukaryotic conserved protein, whose role in translation is poorly understood. Despite intensive study of reading frame maintenance, this entire system involving Asc1, Mbf1, and Rps3, which is specific to eukaryotes, has never been studied. Additional preliminary results have implicated two other proteins in reading frame maintenance: eS26 and Gcn1. Ribosomal protein eS26 sits at the interface of collided ribosomes, which have recently been implicated in frameshifting. Gcn1 is a major regulator of a conserved stress response pathway, involved in sensing uncharged tRNA at the A-site of the ribosome when ribosomes are stalled due to amino acid starvation. Remarkably, three of the twelve most inhibitory codon pairs respond to the RQC system and require Mbf1 for reading frame maintenance, and nine other inhibitory codon pairs do not. The mechanisms by which these nine pairs exert their effects on translation are a mystery, but seem likely to involve central components of the translational control systems as many of these pairs are highly conserved and slowly translated. To follow up on these results we propose to 1. Determine the mechanisms by which Mbf1, Rps3 and Asc1 work to maintain the reading frame. 2. Investigate the roles of Rps26 and Gcn1 proteins in frameshifting. 3. Define the mechanisms by which distinct inhibitory codon pairs exert their effects.