Expanding The Genetic Code In Yeast

大自然允许遗传密码的扩展，包括两种不同寻常的氨基酸：硒半胱氨酸(SEC)和吡咯赖氨酸。然而，有趣的是，这两种独特的氨基酸都没有被真菌利用，从而使酵母成为基因上可操纵的空白石板，用于重建遗传密码扩展。这项建议侧重于这样一个假设，即在酵母中重组SEC掺入将极大地增加我们对SEC掺入机制的理解，并允许操纵这一系统来生产位置特异标记的蛋白质。将UGA终止密码子转化为SEC密码子需要利用一种新的翻译延伸因子(EEFSec)、位于硒蛋白mRNAs 3‘非翻译区的硒半胱氨酸插入序列(SECIS)元件以及一种名为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。这一过程是生产25种人类硒蛋白所必需的，其中许多形成了对抗氧化应激的基本防线。为了在酵母中重建SEC掺入系统，我们建议创造一系列菌株，以允许逐步重建。这一过程将从SEC-tRNASec开始，直到将SEC掺入荧光素酶报告程序，最后是一系列基因筛选，旨在识别增强SEC掺入的因素，并选择能够支持非天然(如荧光)氨基酸的位点特异性掺入的成分。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。 与公众健康相关：该项目旨在重建酿酒酵母中硒半胱氨酸形式的硒的利用。利用硒半胱氨酸所需的独特分子机制将被操纵，以实现蛋白质工程。此外，该系统将成为治疗药物的试验台，这些药物旨在调节体内含硒蛋白质的生产。

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.