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Regulation of Processive Selenocysteine Incorporation in Selenoprotein P

by
Malavika Gupta
A.B., 2000
Washington University
St. Louis

Thesis Advisor: Paul Copeland, PhD
Graduate Program in Molecular Genetics and Microbiology


RWJMS Research Tower
V-10

Tuesday, October 28, 2008
2:00 pm


Abstract

A SECIS element in the 3’ untranslated region and an in-frame UGA codon are the requisite cis -acting elements for the incorporation of selenocysteine into selenoproteins. Equally important are the trans-acting factors SBP2, Sec-tRNA [Ser]Sec, and eEFSec. Multiple in-frame UGAs and two SECIS elements make the mRNA encoding Selenoprotein P (Sel P) unique. We studied the role of codon context in determining the efficiency of UGA readthrough at each of the ten rat Sel P Sec codons and found that in rabbit reticulocyte lysate, differences between the contexts were dramatically reduced in the presence of excess SBP2. Mutational analysis of the “fourth base” of contexts 1 and 5 revealed that only the latter followed the established rules for hierarchy of translation termination. In addition, mutations in either or both of the Sel P SECIS elements resulted in differential effects on UGA readthrough. We also show a competition between SBP2 and G418 for access to the ribosomal A-site and finally, we found that eRF1 mediated translation termination could not override the ability of SBP2 to stimulate Sec incorporation. Interestingly, we also found that endogenous SBP2 in rat testes S17 extract does not bind to the full-length Sel P UTR. To identify other cis elements in the Sel P 3’UTR that may be regulating SBP2 binding and thus Sec incorporation, we generated Sel P 3’UTR truncation and deletion mutants. UV-cross linking of Sel P 3’UTR truncation mutants to rat testes extract allowed us to identify two regions in the Sel P 3’UTR that regulate SBP2. One region lies upstream of SECIS 1 and the other lies upstream of SECIS 2. Furthermore, we have detected proteins that bind specifically to the sequence upstream of SECIS 1 and we have been able to correlate binding of these proteins to the lack of SBP2 binding to the Sel P 3’UTR. Based on 35S-Met incorporation in an in vitro translation assay, we are able to study translation efficiency as well as Sec incorporation as a function of the Sel P 3’UTR. We conclude that a large codon context forms a cis-element that works together to determine readthrough efficiency and that a region upstream of SECIS 1 is involved in regulating translation and thus probably processive Sec incorporation.


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