Expanding The Genetic Code In Yeast

扩展酵母中的遗传密码

• 批准号: 8710794
• 负责人: PAUL R COPELAND
• 金额: $ 2.78万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 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/8710794
• 关键词: Expanding; Genetic; Code; Yeast

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.

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，因此使酿酒酵母成为可遗传操作的空白石板，用于重建遗传密码扩展。该提案的重点是这样的假设：在酵母中重建 Sec 掺入将极大地增加我们对 Sec 掺入机制的理解，并允许操纵该系统来生产位点特异性标记的蛋白质。将 UGA 终止密码子转化为 Sec 密码子需要利用新型翻译延伸因子 (eEFSec)、硒蛋白 mRNA 3' 非翻译区中的硒代半胱氨酸插入序列 (SECIS) 元件以及称为 SBP2 的新型 SECIS 结合蛋白。这些因素协同作用，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 25 种人类硒蛋白生产所必需的，其中许多硒蛋白构成了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，以允许逐步重建。该过程将从 Sec-tRNASec 开始，逐步将 Sec 整合到荧光素酶报告基因中，最后进行一系列遗传筛选，旨在识别增强 Sec 整合的因素，并选择能够支持 Sec 的位点特异性整合的组件。非天然（例如荧光）氨基酸。除了对硒生物学产生重大影响外，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。