TRANSLATIONAL AMBIGUITY IN YEAST

蛋白质合成具有高度的精确度，这取决于 核糖体蛋白等众多成分，以及标准和新的 翻译因素。酵母中全能抑制作用的研究 酿酒酵母是最富有成效的方法之一 找出翻译歧义和发现歧义的因素 真核细胞翻译机制的新组成部分。 此外，酿酒酵母代表了一种特别方便的 研究真核生物翻译机构的模式生物 因为在酵母中发现的基因通常在进化上是保守的 在其他真核生物中也有对应的基因。另外， 强大的基因和DNA重组方法可用于酵母 方便地破坏和修改基因。 拟议研究项目的主要目的是调查 全能抑制子编码的蛋白质的功能 关于SUP45和SUP35，并确定它们在翻译过程中的作用。 Sup45和Sup35蛋白将被提纯并用于无细胞酵母 翻译系统及其与氨基酰基相互作用的研究- TRNA、GTP和mRNA。突变、缺失与寡核苷酸导向 我们将利用突变技术来研究其结构与功能的关系。 这些蛋白质。翻译过程中的具体作用及其意义 与平移设备的其他组件的交互作用将是 用无细胞系统进行了研究。与蛋白质相互作用的研究 Sup45和Sup35蛋白将被用来尝试定义 酵母中翻译与其他过程的相互关系。因为 SUP35和SUP45基因似乎影响不同的细胞过程 包括呼吸和细胞周期控制，我们计划研究核 Sup35和Sup45蛋白的细胞分级和定位 通过间接免疫荧光的方法，研究其可能存在的 这些蛋白质与DNA的相互作用。相应的野生型基因 到新分离的SUP42和SUP43，可能还有其他万能 抑制者，将被测序。如果它们看起来是新奇的翻译 因素，它们的作用将通过这些方法进一步研究 如上所述。质粒介导的野生型基因的扩增， 导致无所不能的镇压，将被用作 识别涉及到的翻译设备的新部件 在真核生物中保持翻译的准确性。

Protein synthesis occurs with a high degree of precision that depends on numerous components, such as ribosomal proteins, and standard and novel translation factors. The study of omnipotent suppression in the yeast Saccharomyces cerevisiae is one of the most fruitful approaches for identifying factors involved in translational ambiguity and for uncovering new components of the translational machinery of eukaryotic cells. Furthermore, the yeast S. cerevisiae represents an especially convenient model organism for investigating the translational apparatus of eukaryotes because the genes identified in yeast are usually evolutionarily conserved and have their counterparts in other eukaryotic organisms. Also, the powerful genetic and DNA recombinant methods available for yeast allows convenient disruption and modification of genes. The main aim of the proposed research project is to investigate the functions of proteins encoded by omnipotent suppressors, with an emphasis on SUP45 and SUP35, and to define their roles in the translational process. the Sup45 and Sup35 proteins will be purified and used in yeast cell-free translational systems and for studies of their interactions with aminoacyl- tRNA, GTP and mRNA. Mutations, deletions and oligonucleotide-directed mutagenesis will be used to study the structure-function relationship of these proteins. The specific role in the translational process and their interaction with other components of the translational apparatus will be investigated with cell-free systems. Studies of proteins interacting with the Sup45 and Sup35 proteins will be used as an attempt to define the interrelationships of translation with other processes in yeast. Because the SUP35 and SUP45 genes appear to affect different cellular processes including, respiration and cell cycle control, we plan to study the nuclear localization of the Sup35 and Sup45 proteins by cellular fractionation and by the method of indirect immunofluorescence, and to study of possible interaction of these proteins with DNA. The wild-type genes corresponding to the newly isolated SUP42 and SUP43, and possibly other omnipotent suppressors, will be sequenced. If they appear to be novel translational factors, their function will be further investigated by the approaches described above. Plasmid-mediated amplification of the wild-type genes, leading to omnipotent suppression, will be used as an approach for identification of novel components of translational apparatus involved in maintaining the translational accuracy in eukaryotes.