Modulation of translation by synonomous codons in yeast

酵母中同义密码子对翻译的调节

基本信息

批准号：
10655468
负责人：
Elizabeth Joan Grayhack
金额：
$ 32.34万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10655468
关键词：
Affect Affinity Chromatography Amino Acids Ataxia Binding Proteins Cells Chimeric Proteins Codon Nucleotides Complex Defect Elements Eukaryota Gene Order Genes Genetic Genetic Code Health Human Intellectual functioning disability Learning Location Maintenance Mass Spectrum Analysis Mediating Messenger RNA Methods Monitor Mutation N-terminal Nerve Degeneration Organism Pathway interactions Peptides Polyribosomes Process Protein Biosynthesis Protein Engineering Proteins Quality Control RPS3 gene Reading Frames Regulation Reporter Ribosomal Frameshifting Ribosomal Proteins Ribosomes Role Saccharomyces cerevisiae Site Starvation System Testing Transfer RNA Translating Translational Repression Translations Variant Work Yeasts biological adaptation to stress crosslink follow-up genetic selection high throughput screening human disease interest mRNA Decay mutant nuclease polypeptide prevent protein aminoacid sequence recruit response role model translation factor translational genetics

项目摘要

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.

项目概要遗传密码从 mRNA 翻译成蛋白质最终决定了蛋白质的组成细胞。翻译延伸及其保真度对于人类健康至关重要，因为翻译因子的突变可导致智力障碍。翻译延伸通过同义密码子的选择来调节用于编码多肽并受到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 密码子对的翻译强烈抑制性，比任何单一密码子都强得多。抑制作用是由核糖体蛋白 Asc1（人 RACK1），当核糖体碰撞时，它会触发核糖体质量控制（RQC）系统的参与。到定义密码子介导的翻译效应的范围和机制，我们最近使用了高 GFP 变体的通量测定可鉴定 17 个强抑制密码子对，其中 12 个属于酵母中翻译最慢的密码子对。我们推断这些对在功能上很重要，因为缓慢翻译的配对在密切相关的物种中基因的相应位置上高度保守。我们也在研究阅读框维护的关键流程，这是阅读框最基本的功能之一。核糖体。我们发现缺乏 Asc1 的菌株在 CGA 密码子重复序列处经历广泛的移码。通过基因选择，我们最近发现了另外两种蛋白质，它们与 Asc1 一起作用以防止 CGA 重复序列的移码：uS3/Rps3（一种普遍保守的核糖体蛋白）和 Mbf1（一种古菌/真核保守蛋白，其在翻译中的作用知之甚少。尽管刻苦学习阅读框维护，整个系统涉及Asc1、Mbf1和Rps3，这是特定于真核生物，从未被研究过。其他初步结果表明另外两种蛋白质参与阅读框维护：eS26 和 GCN1。核糖体蛋白 eS26 位于碰撞核糖体的界面，最近已被证实在移码中。 Gcn1 是保守应激反应通路的主要调节因子，参与传感当核糖体因氨基酸饥饿而停滞时，核糖体 A 位点不带电 tRNA。值得注意的是，十二个最具抑制性的密码子对中的三个对 RQC 系统有反应并需要 Mbf1 用于阅读框维护，而其他九个抑制密码子对则不然。这些机制通过九对对翻译的影响是一个谜，但似乎可能涉及翻译的核心组成部分翻译控制系统，因为其中许多对是高度保守的并且翻译缓慢。为了跟进这些结果，我们建议 1. 确定 Mbf1、Rps3 和 Asc1 的机制努力维持阅读框架。 2. 研究Rps26和Gcn1蛋白在移码中的作用。 3. 定义不同抑制密码子对发挥作用的机制。