Modulation of translation by synonomous codons in yeast

项目摘要 基因密码从信使核糖核酸到蛋白质的翻译最终决定了蛋白质的组成 牢房。翻译延伸性及其保真度对人类健康至关重要，因为翻译因素中的突变 会导致智力残疾。翻译延伸受同义密码子选择的影响 用于编码多肽，并受多种质量控制机制的约束，以防止合成 异常蛋白质。在酿酒酵母中，CGA-CGA密码子对的翻译能力很强 抑制性，比任何单一密码子都要强得多。抑制是由核糖体蛋白Asc1(人 RACK1)，当核糖体碰撞时，它触发核糖体质量控制(RQC)系统的参与。至 定义密码子对翻译影响的范围和机制，我们最近使用了一个很高的 通过对GFP变异体的吞吐能力分析来鉴定17个强抑制密码子对，其中12个属于 在酵母中翻译最慢的密码子对。我们推断这些配对在功能上是重要的，因为 在亲缘关系密切的物种中，缓慢翻译的对在基因的相应位置高度保守。 我们也在研究阅读框维护的关键过程，这是最基本的功能之一 核糖体。我们已经发现，缺乏Asc1的菌株在CGA密码子重复序列上经历了广泛的框架移位。 通过基因选择，我们最近确定了另外两种与Asc1一起工作的蛋白质，以防止 CGA处的移码重复：US3/RPS3，一种普遍保守的核糖体蛋白，以及Mbf1，An。 古细菌/真核生物保守的蛋白质，其在翻译中的作用尚不清楚。尽管进行了密集的研究 在读帧维护中，整个系统涉及Asc1、Mbf1和RPS3，这是特定于 真核生物，从来没有被研究过。 更多的初步结果表明，还有另外两种蛋白质与阅读框架的维持有关：ES26和 Gcn1。核糖体蛋白eS26位于相互碰撞的核糖体的交界处，这是最近发现的 在移帧方面。Gcn1是保守的应激反应途径的主要调节者，参与感知 当核糖体因氨基酸饥饿而停滞时，核糖体A位不带电荷的tRNA。 值得注意的是，12个最具抑制性的密码子对中有3个响应RQC系统并需要Mbf1 对于阅读框的维持，其他9个抑制性密码子对则不能。它们所依据的机制 九对在翻译中发挥作用是一个谜，但似乎涉及到翻译的核心组成部分 翻译控制系统，就像许多这样的配对一样，高度保守，翻译速度很慢。 为了跟进这些结果，我们建议1.确定Mbf1、RPS3和Asc1 努力维护阅读框架。2.研究Rps26和Gcn1蛋白在移码中的作用。3. 定义不同的抑制性密码子对发挥作用的机制。

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.