Functional analysis of SBP2 and selenocysteine incorporation

膳食硒作为氨基酸硒代半胱氨酸（Sec）被掺入至少25种人类蛋白质中。 在延伸多肽中的Sec掺入代表了标准蛋白质合成多肽的修饰。 这是因为它需要利用一种新的翻译延伸因子（eEFSec），一种硒代半胱氨酸， 插入序列（SECIS）元件在3'非翻译区的硒蛋白mRNA，和一个新的 SECIS结合蛋白，称为SBP 2。这些因素协同作用，改变特定的编码潜力， UGA密码子通过指定Sec特异性tRNA的插入，Sec-tRNA[Ser]Sec.的重点 这是关于SBP 2及其作用机制的建议。迄今为止，功能分析已经确定，SBP 2 是Sec掺入所必需的，具有特异性SECIS元件结合活性， 与核糖体相互作用。SBP 2的结构/功能分析表明，它由三个组成， 独特的结构域：没有已知功能的JNK-末端结构域， 是Sec掺入所需的，但不是SECIS元件结合所需的，并且C-末端SECIS元件结合 含有在扭结-转角结合蛋白家族中发现的RNA结合基序的结构域（例如核糖体 蛋白L7 Ae）。使用体外研究和基于细胞的测定的组合，所提出的实验是 旨在破译SBP 2亚结构域内的结构/功能关系，并鉴定新的 使用三层方法的证券公司注册机制的组成部分。首先，我们建议 精确定义Sec掺入所需的氨基酸，以便为结构分析奠定坚实的基础。 问题研究其次，我们将开发分析方法来研究SBP 2 N末端结构域的功能，以便 深入了解其在证券交易委员会成立中的潜在监管作用。第三，我们将确定 通过组装以SBP 2为中心和以硒蛋白mRNA为中心的Sec掺入复合物（SIC） 哺乳动物细胞中的复合物，随后进行复合物纯化和鉴定。总的来说，这项工作将 提供有关证券公司注册机制的基本和必要信息- 这一进程将成为旨在最大限度地发挥 硒蛋白功能

Dietary selenium is incorporated into at least 25 human proteins as the amino acid selenocysteine (Sec). Sec incorporation in an elongating polypeptide represents a modification of the standard 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 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-tRNA[Ser]Sec. The focus of this proposal is on SBP2 and its mechanism of action. To date, functional analyses have established that SBP2 is required for Sec incorporation, possesses specific SECIS element binding activity and also physically interacts with the ribosome. Structure/function analysis of SBP2 has shown that it is comprised of three distinct domain: a dispensable N-terminal domain with no known function, a central "functional domain" that is required for Sec incorporation but not SECIS element binding, and a C-terminal SECIS element binding domain containing an RNA binding motif found in the family of kink-turn binding proteins (e.g.ribosomal protein L7Ae). Using a combination of in vitro studies and cell-based assays, the experiments proposed are designed to decipher the structure/function relationships within the SBP2 subdomains and identify novel components of the Sec incorporation machinery using a three-tiered approach. First, we propose to precisely define the amino acids required for Sec incorporation in order to lay a solid foundation for structural studies. Second, we will develop assays to study the function of the SBP2 N-terminal domain in order to gain insight into its potential regulatory role in Sec incorporation. Third, we will identify components of the Sec incorporation complex (SIC)by assembling SBP2-centered and selenoprotein mRNA-centered complexes in mammalian cells followed by complex purification and identification. As a whole, this work will provide fundamental and essential information regarding the mechanism of Sec incorporation - an essential process that will be an important target for strategies designed to maximize the beneficial properties of selenoprotein function.