title: Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination.
abstract: The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.
authors.affiliation: Posttranscriptional Control Group, Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Manchester M60 1QD, United Kingdom.
authors.forename: M T
authors.initials: MT
authors.lastname: Nasim
authors.forename: S
authors.initials: S
authors.lastname: Jaenecke
authors.forename: A
authors.initials: A
authors.lastname: Belduz
authors.forename: H
authors.initials: H
authors.lastname: Kollmus
authors.forename: L
authors.initials: L
authors.lastname: Flohé
authors.forename: J E
authors.initials: JE
authors.lastname: McCarthy
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon / Q000235:genetics; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010443:Peptide Chain Termination, Translational; D010957:Plasmids; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D017279:Selenocysteine / Q000378:metabolism*; D051140:Selenoproteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014981:Xenopus
chemical_list: D020413:3' Untranslated Regions; D003062:Codon; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D012345:RNA, Transfer, Amino Acid-Specific; D051140:Selenoproteins; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D003545:Cysteine