The translational control of selenoprotein synthesis

硒蛋白合成的翻译控制

• 批准号: 6755934
• 负责人: PAUL R COPELAND
• 金额: $ 32.51万
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6755934
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; affinity chromatography; binding proteins; gel filtration chromatography; genetic translation; nucleic acid sequence; polymerase chain reaction; protein biosynthesis; ribosomal RNA; selenoprotein; western blottings

DESCRIPTION (provided by applicant): Selenocysteine is a unique amino acid that is incorporated specifically into a select group of essential proteins. This process represents a modification of the standard eukaryotic protein synthetic machinery in that it requires the utilization of a novel translation elongation factor (eEFSec), a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region (UTR) of selenoprotein mRNAs, and a novel SECIS binding protein termed SBP2. These factors act in concert to alter the coding potential of specific in-frame stop codons (UGA) by specifying the insertion of selenocysteine at that site. The goal of this project is to understand the interplay between SBP2 and the translation machinery. Since SBP2 is a limiting factor required for Sec incorporation, it is central to the regulation of selenoprotein synthesis, and many selenoproteins are known to play critical roles in cellular defense from oxidation. As a result, SBP2 will be a key target for methods designed to increase the beneficial properties of selenoproteins including the prevention of DNA damage in carcinogenesis and oxidation of pathogenic lipids involved in atherosclerosis. This proposal seeks to understand the function of SBP2 by studying its specific interaction with ribosomes and its interaction with the machinery that would ordinarily dictate the termination of translation at selenocysteine (UGA) codons. Our preliminary studies indicate that SBP2 specifically interacts with ribosomal RNA from yeast to rodents, indicating that the SECIS element may have evolved from highly conserved rRNA structures. This study will combine the utilization of mammalian cells as well as the yeast S. cerevisiae in order to explore this interaction while exploiting the unique benefits of each system. Specifically, we will use mammalian rRNA fragments to probe the binding characteristics of SBP2. This will be integrated with an analysis of SBP2 binding to intact ribosomes using a system unique to yeast by which homogeneous mutant ribosomes can be produced in vivo. Based on the dual function of the UGA stop codon, we will directly assess the role of SBP2 in preventing termination at selenocysteine codons by analyzing selenocysteine incorporation from efficient and inefficient templates. The information we obtain about the function of SBP2 will be used to develop a detailed model for selenocysteine incorporation which should shed light on the basic mechanism of translation termination as well as enable us to identify components of the system that can be manipulated in order to optimize selenoprotein synthesis.

描述（由申请人提供）： 硒代半胱氨酸是一种独特的氨基酸，专门掺入了一组必需蛋白中。该过程代表了标准真核蛋白合成机制的修改，因为它需要使用新型翻译伸长因子（EEFSEC），这是3'未经翻译的3'未经翻译区域（UTR）的硒代半胱氨酸插入序列（SECIS）元素的硒蛋白酶MRNA MRNA MRNA和新型Secis secis bitiS bitect bitting Sbpppppppppppppppppppppppp，这些因素协同起作用，以通过指定在该站点的插入硒代海囊素来改变特定框架终止密码子（UGA）的编码潜力。该项目的目的是了解SBP2和翻译机械之间的相互作用。由于SBP2是SEC掺入所需的限制因素，因此它对于硒蛋白合成的调节至关重要，并且已知许多硒蛋白在氧化中起关键作用。结果，SBP2将是旨在提高硒蛋白的有益特性的方法的关键目标，包括预防癌变中的DNA损伤以及涉及动脉粥样硬化的致病性脂质的氧化。该提案试图通过研究其与核糖体的特定相互作用以及与机械的相互作用来理解SBP2的功能，该机械通常决定在硒代型（UGA）密码子上终止翻译。我们的初步研究表明，SBP2特异性与从酵母到啮齿动物的核糖体RNA相互作用，表明SECIS元素可能是从高度保守的RRNA结构中演变而成的。这项研究将结合哺乳动物细胞和酵母菌的利用率，以探索这种相互作用，同时利用每个系统的独特益处。具体而言，我们将使用哺乳动物rRNA片段来探测SBP2的结合特征。这将与SBP2结合到完整核糖体的分析，使用酵母独有的系统在体内产生均匀的突变核糖体。基于UGA终止密码子的双重函数，我们将直接评估SBP2通过分析有效且效率低下的模板中的硒代型半胱氨酸掺入来防止硒代码密码子终止的作用。我们获得的有关SBP2功能的信息将用于开发用于硒吻氨酸融合的详细模型，该模型应阐明转换终止的基本机制，并使我们能够识别可以操纵的系统组件以优化硒蛋白合成的系统。