Molecular genetics of translational accuracy

核糖体上的蛋白质合成过程是最古老的生物机制之一，具有 可能是在第一个细胞之前的RNA世界中进化的。核糖体进化出一种精细的结构， 生命的各个领域都保存着精华的部分。了解核糖体的作用机制 将mRNA忠实地翻译成蛋白质已经变得更加重要，也更容易处理 细菌核糖体两个亚基的分子结构和低分辨结构的解 酵母核糖体。 我们的实验室多年来一直对mrna序列如何与之相互作用的问题感兴趣。 核糖体导致程序性错误。这些错误包括程序化的平移框架， 核糖体的哪些靶位元件参与维持保真度以使核糖体发生移位 以接近100%的频率读取帧。随机位置上的正弦移码发生的几率要小得多 每20000个密码子中就有一个，当地保真度的变化真的是巨大的。 蛋白质合成的准确性包括接受正确的(同源的)氨基酰-tRNA结合 延伸因子1A(EF-1A)进入核糖体A位点，并拒绝错误的(非同源的)。 区分包括动态校对和诱导匹配，在这两种情况下，同源AA-tRNA增加了 通过诱导核糖体的结构变化，它们自身被接受的可能性。我们不明白 这种结构性变化的性质或其监管。Venki Ramakrishnan提出了这一部分 机制涉及破坏核糖体蛋白S4(RPS4)和RPS4之间的蛋白质-蛋白质相互作用 RpS5。打破这种相互作用可能会导致核糖体“关闭”，将tRNA捕获在A位点和 允许退出EF-1A-GDP。该模型的证据包括少量容易出错的突变 这会改变界面内和界面附近的残留物。我们建议通过以下方式更严格地检验这一假设 在RPS4-rpS5界面和潜在的相互作用途径中发现更多容易出错的突变 在从解码位置引出的核糖体中。这项工作将在细菌和酵母中完成。我们会 通过研究精确度效应，研究哪些反式作用蛋白与精确度机制相互作用 酵母中的一类候选基因缺失。

The process of protein synthesis on the ribosome is among the oldest of biological mechanisms, having evolved probably in the RNA world before the first cells. The ribosome has evolved an elaborate structure, the essential parts conserved in all domains of life. Understanding the mechanism by which the ribosome faithfully translates mRNAs into proteins has become both more important and more tractable with the solution of molecular structures of both subunits of bacterial ribosomes and a lower resolution structure of the yeast ribosome. Our laboratory for many years has been interested in the problem of how mRNA sequences interact with the ribosome to cause programmed errors. These errors include programmed translational frameshifts, which target elements of the ribosome involved in maintaining fidelity in order to cause the ribosome to shift reading frames at frequencies approaching 100%. Sine e frameshifting at random sites occurs at much less than one in 20,000 codons, the local change in fidelity is truly immense. Accuracy in protein synthesis consists of accepting the correct (cognate) aminoacyl-tRNAs bound to elongation factor 1A (EF-1A) into the ribosomal A site and rejecting incorrect (non-cognate) ones. Discrimination involves both kinetic proofreading and induced fit in which cognate aa-tRNAs increase the probability of their own acceptance by inducing a structural change in the ribosome. We do not understand the nature of this structural change or its regulation. Venki Ramakrishnan has proposed that part of the mechanism involves destabilizing a protein-protein interaction between ribosomal proteins S4 (rpS4) and rpS5. Breaking this interaction may cause the ribosome to "close", trapping the tRNA in the A site and allowing exit of EF-1A-GDP. The evidence for the model consists of a small number of error-prone mutations that alter residues in and near the interface. We propose to test this hypothesis more stringently by identifying more erro-prone mutations both in the rpS4-rpS5 interface and in a potential interaction pathway in the ribosome leading from the decoding sites. This work will be done both in bacteria and in yeast. We will also study which trans-acting proteins interact with this accuracy mechanism by studying the accuracy effect of a class of candidate gene deletions in yeast.