The selenocysteine-specific elongation factor contains a novel and multi-functional domain

Abstract

The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNASec) 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 high-lighted 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: GT Phydrolysis, Sec-tRNASec binding, and SBP2/SECIS binding. In addition, wildtype 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-tRNASec binding and accommodation into the ribosomal A-site. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.