STRUCTURE AND FUNCTION OF THE SELENIUM TRANSLATION ELEMENT IN THE 3'-UNTRANSLATED REGION OF HUMAN CELLULAR GLUTATHIONE-PEROXIDASE MESSENGER-RNA

In eukaryotes, incorporation of selenocysteine into the polypeptide ch ain at a UGA codon requires a unique sequence motif, or ''selenium tra nslation element'' (STE), located in the 3'-untranslated region of the mRNA. The present study examines structure-function relationships of conserved sequence elements and of the putative stem-loop secondary st ructure in the STE of human GPX1 mRNA, which encodes the important ant ioxidant enzyme cellular glutathione peroxidase (EC 1.11.1.9). Deletio n of the basal stem, upper stem, or apical loop of the stem-loop struc ture eliminated the ability of the SIE to direct selenocysteine incorp oration at the UGA codon of an epitope-tagged GPX1 reporter construct transfected into COS1 cells. However, mutations that change the primar y nucleotide sequence of nonconserved portions of the stem-loop, but p reserve its overall secondary structure, by inversion of apical loop s equences or exchange of 5' and 3' sides of stem segments, had little o r no effect on selenocysteine incorporation. Effects of single- and do uble-nucleotide substitutions in three short, highly conserved element s in the GPX1 STE depended in large part on their computer-predicted p erturbation of the stem-loop and its midstem bulge. Only in the conser ved ''AAA'' apical loop sequence did mutations show major effects on f unction without predicted changes in secondary structure. Our results demonstrate the critical role of the three short, highly conserved seq uences. However, outside of these elements, the function of the human GPX1 STE appears to depend strongly on the stem-loop secondary structu re.