Processive selenocysteine incorporation during synthesis of eukaryotic selenoproteins.
Selenoproteins are a family of proteins that share the common feature of containing selenocysteine, the "twenty-first" amino acid. Selenocysteine incorporation occurs during translation of selenoprotein messages by redefinition of UGA codons, which normally specify termination of translation. Studies of the eukaryotic selenocysteine incorporation mechanism suggest that selenocysteine insertion is inefficient compared with termination. Nevertheless, selenoprotein P and several other selenoproteins are known to contain multiple selenocysteines. The production of full-length (FL) protein from these messages would seem to demand highly efficient selenocysteine incorporation due to the compounding effect of termination at each UGA codon. We present data demonstrating that efficient incorporation of multiple selenocysteines can be reconstituted in rabbit reticulocyte lysate translation reactions. Selenocysteine incorporation at the first UGA codon is inefficient but increases by approximately 10-fold at subsequent downstream UGA codons. We found that ribosomes in the "processive" phase of selenocysteine incorporation (i.e., after decoding the first UGA codon as selenocysteine) are fully competent to terminate translation at UAG and UAA codons, that ribosomes become less efficient at selenocysteine incorporation as the distance between UGA codons is increased, and that efficient selenocysteine incorporation is not dependent on cis-acting elements unique to selenoprotein P. Furthermore, we found that the percentage of ribosomes decoding a UGA codon as selenocysteine rather than termination can be increased by 3- to 5-fold by placing the murine leukemia virus UAG read-through element upstream of the first UGA codon or by providing a competing messenger RNA in trans. The mechanisms of selenocysteine incorporation and selenoprotein synthesis are discussed in light of these results.
Fixsen
S M
SM
Department of Human Genetics, University of Utah, Room 2100, 15 North 2030 East, Salt Lake City, UT 84112, USA.
Howard
Michael T
MT
D000818:Animals; D003062:Codon; D018388:Codon, Terminator; D066298:In Vitro Techniques; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D017279:Selenocysteine / Q000235:genetics / Q000378:metabolism*; D051149:Selenoprotein P / Q000096:biosynthesis / Q000235:genetics; D051140:Selenoproteins / Q000096:biosynthesis* / Q000235:genetics
D003062:Codon; D018388:Codon, Terminator; D051149:Selenoprotein P; D051140:Selenoproteins; D017279:Selenocysteine