Functional analysis of SBP2 and selenocysteine incorporation

膳食中的硒以硒半胱氨酸(Sec)的形式存在于至少25种人体蛋白质中。 在细长多肽中掺入SEC代表对标准蛋白质合成的修饰 因为它需要使用一种新的翻译延长因子(EEFSec)，即硒半胱氨酸 硒蛋白mRNAs 3‘非翻译区的插入序列(SECIS)元件和一个新的 SECIS结合蛋白称为SBP2。这些因素共同作用，改变了特定基因的编码潜力 UGA密码子通过指定SEC特定的tRNA的插入，SEC-tRNA[Ser]SEC。这件事的重点是 建议是关于SBP2及其作用机制的。迄今为止，功能分析已经证实SBP2 是SEC合并所必需的，具有特定的SECIS元件结合活性，还具有物理上的 与核糖体相互作用。SBP2的结构/功能分析表明，它由三个部分组成 不同的结构域：一个没有已知功能的可有可无的N-末端结构域，一个中心的“功能结构域” 是SEC掺入所必需的，但不是SECIS元件结合所必需的，以及C-末端SECIS元件结合 包含在扭结-转弯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L7Ae)。利用体外研究和基于细胞的分析相结合的方法，提出的实验是 旨在破译SBP2亚域内的结构/功能关系并识别新的 使用三层方法的证券交易委员会成立机制的组成部分。首先，我们建议 准确定义SEC掺入所需的氨基酸，为结构分析奠定坚实基础 学习。其次，我们将开发各种方法来研究SBP2 N-末端结构域的功能，以便 洞察其在SEC注册过程中的潜在监管角色。第三，我们将确定 以SBP2为中心和硒蛋白为中心的SEC掺入复合体(SIC) 哺乳动物细胞中的复合体，然后进行复合体的纯化和鉴定。作为一个整体，这项工作将 提供有关证券交易委员会注册机制的基本和基本信息-基本信息 这一过程将成为旨在最大限度地提高 硒蛋白的功能。

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.