TRANSLATIONAL AMBIGUITY IN YEAST

蛋白质合成的精确度很高，这取决于 许多成分，例如核糖体蛋白，以及标准和新颖的 翻译因素。 酵母全能抑制的研究 酿酒酵母是最富有成效的方法之一 识别涉及翻译歧义的因素并揭示 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 研究真核生物翻译装置的模型生物 因为在酵母中发现的基因通常在进化上是保守的 并在其他真核生物中也有其对应物。 另外， 可用于酵母的强大遗传和 DNA 重组方法允许 方便地破坏和修饰基因。 拟议研究项目的主要目的是调查 由万能抑制因子编码的蛋白质的功能，重点是 SUP45 和 SUP35，并定义它们在翻译过程中的角色。 Sup45 和 Sup35 蛋白将被纯化并用于无细胞酵母 翻译系统及其与氨酰基相互作用的研究 tRNA、GTP 和 mRNA。 突变、缺失和寡核苷酸定向 诱变将用于研究结构与功能的关系 这些蛋白质。 翻译过程中的具体作用及其 与翻译装置的其他组件的相互作用将是 用无细胞系统进行研究。 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 通过细胞分级分离 Sup35 和 Sup45 蛋白的定位 通过间接免疫荧光的方法，研究可能的 这些蛋白质与 DNA 的相互作用。 野生型基因对应 到新分离的 SUP42 和 SUP43，以及可能其他万能的 抑制器，将被测序。 如果它们看起来是新颖的翻译 因素，它们的功能将通过这些方法进一步研究 如上所述。 野生型基因的质粒介导的扩增， 导致全能镇压，将被用作一种方法 识别涉及的翻译装置的新组件 维持真核生物的翻译准确性。

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.