Modulation of translation by synonymous codons in yeast

 描述(由申请人提供)：将基因密码从信使核糖核酸翻译成蛋白质，最终决定细胞的蛋白质组成。在所有三个王国中，翻译效率和保真度都受到用于编码多肽的同义密码子的选择的影响，尽管同义密码子指定插入相同的氨基酸。密码子选择如何影响翻译是生物学的核心问题，但在一定程度上很难研究，因为导致翻译减少的密码子或密码子组合的身份在很大程度上是未知的，特别是在真核生物中。为此，我们首次系统分析了导致酿酒酵母翻译效率下降的密码子，发现由于核糖体中的I*A摆动解码相互作用，Arg CGA密码子的翻译效率极低，并且CGA-CGA密码子对比单独的CGA密码子更具抑制性，这意味着存在其他由不相同密码子组成的抑制对。为了识别这种抑制对，我们开发了一种结合荧光报告和深度测序的方法，用来识别14个强抑制密码子对。在研究CGA密码子对的抑制机制时，我们发现缺乏核糖体蛋白Asc1的菌株阅读CGA密码子的效率更高，而且令人惊讶的是，在CGA重复序列上也经历了广泛的移码。它的人类同源基因Rack1在信号通路中起着中心调节作用。我们在这里建议跟进三个目标。首先，我们将确定新发现的14个抑制性密码子对如何损害翻译。为了确定这些机制，我们将研究已知的质量控制途径在这些密码子对抑制中的作用，我们将通过选择具有改进的抑制密码子对解码的突变体来识别参与密码子介导的抑制的新基因，并确定核糖体是否在这些密码子对处停滞。其次，我们建议通过鉴定其他基因的突变来研究Asc1和其他蛋白质在阅读框架维持中的作用，这些基因独立地导致CGA重复序列的移码，或者与Asc1一起参与防止移码。第三，为了评估抑制性密码子对的功能作用(S)，我们将通过比较这些抑制性密码子对在具有与酿酒酵母相似和不同解码策略的物种中的保守性来确定这些密码子对的保守是否与它们的抑制功能有关。我们还将通过检测表达非天然抑制子tRNAs对酵母适合度的影响来评估抑制密码子对功能改变的后果，并通过研究突变其特定抑制密码子对其表达、功能、RNA结构和表达变异性的影响来评估在特定基因中改变抑制密码子对功能的后果。这些目标的完成很有可能导致对核糖体如何调控基因表达这一根本问题的重大洞察，并有望揭示在进化中保守的全球调控机制。

 DESCRIPTION (provided by applicant): Translation of the genetic code from mRNA into protein ultimately determines the protein composition of the cell. In all three kingdoms, translation efficiency and fidelity are modulated by the choice of synonymous codons used to encode polypeptides, although synonymous codons specify insertion of the same amino acid. The problem of how codon choice affects translation is central to biology, but has been difficult to study in part, because the identities of the codons or codon combinations that cause decreased translation have been largely unknown, particularly in eukaryotes. To this end, we performed the first systematic analysis to identify codons that cause decreased translation efficiency in the yeast Saccharomyces cerevisiae, finding that the Arg CGA codon is translated extremely inefficiently, due to I*A wobble decoding interactions in the ribosome, and that CGA-CGA codon pairs are far more inhibitory than isolated CGA codons, implying that other inhibitory pairs composed of non-identical codons exist. To identify such inhibitory pairs, we developed a method coupling fluorescent reporters and deep sequencing, with which we identified 14 strongly inhibitory codon pairs. In examining the mechanism of inhibition by CGA codon pairs, we found that strains lacking the ribosomal protein Asc1 read through CGA codons more efficiently, and also surprisingly undergo extensive frameshifting at CGA repeats. Its human homolog Rack1 has a central regulatory role in signaling pathways. We propose here to follow up with three aims. First, we will determine how the 14 newly identified inhibitory codon pairs impair translation. To define these mechanisms, we will examine the roles of known quality control pathways in inhibition by these pairs, we will identify new genes involved in codon-mediated inhibition by selecting mutants with improved decoding of inhibitory codon pairs, and we will ascertain whether or not ribosomes stall at these codon pairs. Second, we propose to investigate the role of Asc1 and other proteins in reading frame maintenance, by identifying mutations in other genes that independently cause frameshifting at CGA repeats, or that participate with Asc1 in preventing frameshifting. Third, to assess the functional role(s) of inhibitory codon pairs, we will determine if conservation of these codon pairs is related to their inhibitory function by comparing conservation of these inhibitory codon pairs in species with similar and different decoding strategies from S. cerevisiae. We will also evaluate the consequences of altering inhibitory codon pair function, both in the entire organism by examining the effects of expressing non-native suppressor tRNAs on yeast fitness, and in specific genes by investigating effects of mutating their specific inhibitory codon pairs on their expression, function, RNA structure and variability of expression. Completion of these aims is highly likely to result in significant insights into the fundamental question of how ribosomes regulate gene expression, and has high promise for uncovering global regulatory mechanisms that are conserved in evolution.