Expanding The Genetic Code In Yeast

扩展酵母中的遗传密码

• 批准号: 7994428
• 负责人: PAUL R COPELAND
• 金额: $ 30.86万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994428
• 关键词: 3' Untranslated Regions; Adverse effects; Amino Acids; Anabolism; Beds; Binding Proteins; Biology; Code; Codon Nucleotides; Complex; DNA Insertion Elements; Discipline; Elongation Factor; Enhancers; Genetic; Genetic Code; Genetic Screening; Gold; Human; IS Elements; Label; Luciferases; Mammals; Methods; Modification; Molecular; Nature; Oxidative Stress; Pathway interactions; Phase; Positioning Attribute; Process; Production; Protein Biosynthesis; Protein Engineering; Proteins; Reaction; Recombinant Proteins; Reporter; Resources; Saccharomyces cerevisiae; Selenium; Selenocysteine; Series; Site; Specific qualifier value; Specificity; Synthetic Genes; System; Technology; Terminator Codon; Testing; Therapeutic Agents; Transfer RNA; Translation Process; Translations; Vascular Plant; Work; Yeasts; cDNA Library; cis acting element; design; enhancing factor; flexibility; fungus; improved; in vivo; mutant; new technology; novel; plant fungi; promoter; protein structure function; public health relevance; pyrrolysine; reconstitution; selenocysteine-tRNA; selenoprotein

Nature has allowed the expansion of the genetic code to include two unusual amino acids: selenocysteine (Sec) and pyrrolysine. Interestingly, however, neither of these unique amino acids are utilized by fungi, thus making the yeast Saccharomyces cerevisiae a genetically manipulable blank slate for reconstituting genetic code expansion. This proposal focuses on the hypothesis that reconstitution of Sec incorporation in yeast will dramatically increase our understanding of the mechanism of Sec incorporation as well as allow for the manipulation of this system for the production of site-specifically labeled proteins. The transformation of a UGA stop codon into a Sec codon requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific UGA codons by specifying the insertion of the Sec-specific tRNA, Sec-tRNASec. This process is required for the production of 25 human selenoproteins, many of which form an essential line of defense against oxidative stress. In order to rebuild the Sec incorporation system in yeast, we propose to create a series of strains that will allow a stepwise approach to reconstitution. The process will start with Sec- tRNASec, moving up to incorporation of Sec into a luciferase reporter and culminating with a series of genetic screens designed to identify factors that enhance Sec incorporation as well as select for components able to support the site-specific incorporation of unnatural (e.g. fluorescent) amino acids. In addition to the major impact on selenium biology, this project will also provide valuable resources for scientific disciplines that require the analysis of protein structure and function. PUBLIC HEALTH RELEVANCE: This project is designed to reconstitute the utilization of selenium in the form of selenocysteine in the yeast Saccharomyces cerevisiae. The unique molecular machinery that is required for selenocysteine utilization will be manipulated to allow protein engineering. In addition, this system will form the test bed for therapeutics agents designed to regulated the production of selenium-containing proteins in vivo.

大自然允许遗传密码的扩展，包括两种不寻常的氨基酸：硒代半胱氨酸（Sec）和吡咯赖氨酸。然而，有趣的是，这些独特的氨基酸都不被真菌利用，因此使酵母酿酒酵母成为一种可遗传操纵的空白石板，用于重建遗传密码扩增。该建议的重点是假设，即在酵母中重组的Sec掺入将大大增加我们对Sec掺入机制的理解，并允许操纵该系统用于生产位点特异性标记的蛋白质。将UGA终止密码子转化为Sec密码子需要利用新的翻译延伸因子（eEFSec）、硒蛋白mRNA的3'非翻译区中的硒代半胱氨酸插入序列（SECIS）元件和称为SBP 2的新的SECIS结合蛋白。这些因素共同作用，通过指定Sec特异性tRNA（Sec-tRNASec）的插入来改变特定UGA密码子的编码潜力。这一过程是生产25种人类硒蛋白所必需的，其中许多硒蛋白是抵御氧化应激的重要防线。为了重建酵母中的Sec掺入系统，我们建议创建一系列菌株，这些菌株将允许逐步重建的方法。该过程将从Sec-tRNASec开始，直到将Sec掺入荧光素酶报告基因，并以一系列遗传筛选为终点，这些遗传筛选旨在鉴定增强Sec掺入的因子以及选择能够支持非天然（例如荧光）氨基酸的位点特异性掺入的组分。除了对硒生物学的重大影响外，该项目还将为需要分析蛋白质结构和功能的科学学科提供宝贵的资源。 公共卫生相关性：本项目旨在重建酵母中硒以硒代半胱氨酸形式的利用。硒代半胱氨酸利用所需的独特分子机制将被操纵以允许蛋白质工程。此外，该系统将形成用于调节体内含硒蛋白质产生的治疗剂的试验床。