Characterization of canonical and novel functions of the Selenocysteine-specific elongation factor eEFSec
Jonathan N. González-Flores
B.S., University of Puerto Rico, Mayagüez-2007
Thesis Advisor: Paul R. Copeland, Ph.D.
Graduate Program in Molecular Genetics, Microbiology & Immunology
RWJMS Research Tower, Room V-14
Monday, December 17, 2012
The selenocysteine (Sec)-specific eukaryotic elongation factor (eEFSec) delivers the aminoacylated selenocysteine-tRNA (Sec-tRNA[Ser]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[Ser]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[Ser]Sec binding and accommodation into the ribosomal A-site.
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