Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2012 Nov 9;287(46):38936-45. doi: 10.1074/jbc.M112.415463. Epub 2012 Sep 19.
The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA(Sec)) to the ribosome and suppresses UGA codons that are upstream of Sec insertion sequence (SECIS) elements bound by SECIS-binding protein 2 (SBP2). Multiple studies have highlighted the importance of SBP2 forming a complex with the SECIS element, but it is not clear how this regulates eEFSec during Sec incorporation. Compared with the canonical elongation factor eEF1A, eEFSec has a unique C-terminal extension called Domain IV. To understand the role of Domain IV in Sec incorporation, we examined a series of mutant proteins for all of the known molecular functions for eEFSec: GTP hydrolysis, Sec-tRNA(Sec) binding, and SBP2/SECIS binding. In addition, wild-type and mutant versions of eEFSec were analyzed for Sec incorporation activity in a novel eEFSec-dependent translation extract. We have found that Domain IV is essential for both tRNA and SBP2 binding as well as regulating GTPase activity. We propose a model where the SBP2/SECIS complex activates eEFSec by directing functional interactions between Domain IV and the ribosome to promote Sec-tRNA(Sec) binding and accommodation into the ribosomal A-site.
硒代半胱氨酸（Sec）特异性真核延伸因子（eEFSec）将氨酰化的硒代半胱氨酸-tRNA（Sec-tRNA（Sec））递送到核糖体，并抑制 UGA 密码子，这些密码子位于 SECIS 结合蛋白 2（SBP2）结合的 Sec 插入序列（SECIS）元件的上游。多项研究强调了 SBP2 与 SECIS 元件形成复合物的重要性，但尚不清楚这如何在 Sec 掺入过程中调节 eEFSec。与典型的延伸因子 eEF1A 相比，eEFSec 具有独特的 C 末端延伸结构域 IV。为了了解结构域 IV 在 Sec 掺入中的作用，我们研究了一系列突变蛋白，这些蛋白具有 eEFSec 的所有已知分子功能：GTP 水解、Sec-tRNA（Sec）结合和 SBP2/SECIS 结合。此外，还分析了野生型和突变型 eEFSec 在新型依赖于 eEFSec 的翻译提取物中的 Sec 掺入活性。我们发现结构域 IV 对于 tRNA 和 SBP2 结合以及调节 GTPase 活性都是必不可少的。我们提出了一个模型，其中 SBP2/SECIS 复合物通过指导结构域 IV 与核糖体之间的功能相互作用来激活 eEFSec，从而促进 Sec-tRNA（Sec）结合并适应核糖体的 A 位。
