Expanding The Genetic Code In Yeast

大自然允许遗传密码的扩展，包括两种不寻常的氨基酸：硒半胱氨酸（Sec）和吡啶赖氨酸。然而，有趣的是，这两种独特的氨基酸都不被真菌利用，因此，酵母酿酒酵母是一个基因上可操纵的空白石板，用于重建遗传密码扩展。这一建议的重点是假设重组Sec在酵母中的结合将极大地增加我们对Sec结合机制的理解，并允许对该系统进行操作，以生产位点特异性标记的蛋白质。将UGA终止密码子转化为Sec密码子需要利用一种新的翻译延伸因子（eEFSec）、硒蛋白mrna 3'非翻译区中的硒半胱氨酸插入序列（SECIS）元件和一种称为SBP2的新型SECIS结合蛋白。这些因子协同作用，通过指定sec特异性tRNA （Sec-tRNASec）的插入来改变特定UGA密码子的编码潜能。这一过程是人体产生25种硒蛋白所必需的，其中许多硒蛋白构成了抵御氧化应激的重要防线。为了在酵母中重建Sec并入系统，我们建议创建一系列菌株，使其能够逐步重建。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，最后进行一系列基因筛选，旨在识别增强Sec整合的因素，并选择能够支持非天然（如荧光）氨基酸的位点特异性整合的成分。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。

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.