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J. Biol. Chem., Vol. 282, Issue 51, 36797-36807, December 21, 2007

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Functional Analysis of the Interplay between Translation Termination, Selenocysteine Codon Context, and Selenocysteine Insertion Sequence-binding Protein 2^*

From the Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

A selenocysteine insertion sequence (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. To study the role of codon context in determining the efficiency of UGA readthrough at each of the 10 rat Sel P Sec codons, we individually cloned 27-nucleotide-long fragments representing each UGA codon context into a luciferase reporter construct harboring both Sel P SECIS elements. Significant differences, spanning an 8-fold range of UGA readthrough efficiency, were observed, but these differences 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. Interestingly, even when both SECIS elements harbored a mutation of the core region required for Sec incorporation, context 5 retained a significantly higher level of readthrough than context 1. We also show that SBP2-dependent Sec incorporation is able to repress G418-induced UGA readthrough as well as eRF1-induced stimulation of termination. We conclude that a large codon context forms a cis-element that works together with Sec incorporation factors to determine readthrough efficiency.

Received for publication, August 22, 2007 , and in revised form, October 19, 2007.

^* This work was supported by United States Public Health Service Grant GM068077. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: 675 Hoes Lane, Piscataway, NJ 08854. Fax: 732-235-5223; E-mail: paul.copeland{at}umdnj.edu.

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