title: A G.U base pair in the eukaryotic selenocysteine tRNA is important for interaction with SePF, the putative selenocysteine-specific elongation factor.
abstract: In Escherichia coli, selenocysteine biosynthesis and incorporation into selenoproteins requires the action of four gene products, including the specialized selenocysteine tRNA(Sec) and elongation factor SELB, different from the universal EF-Tu. In this regard, the situation is less clear in eukaryotes, but we previously reported the existence of SePF, a putative SELB homologue. The secondary structure of the tRNA(Sec) differs slightly in eukaryotes, due to a change in the lengths of several stems. Two non-Watson-Crick base pairs, G5a x U67b and U6 x U67, reside in the acceptor stem and are conserved in the course of evolution. Since it has already been reported that changing them to Watson-Crick base pairs did not affect the serylation or selenylation levels of tRNA(Sec), we asked whether these non-Watson-Crick base pairs are required for the interaction with SePF. To this end, tRNA(Sec) variants carrying Watson-Crick changes at these positions were tested for their ability to maintain the interaction with SePF. In these assays, the tRNA(Sec)-SePF interaction was determined by the protective action it confers against hydrolysis of the amino acid ester bond, under basic conditions. All the changes introduced at U6 x U67 did not significantly affect the interaction. Interestingly, however, the G5a x U67b to G5a-C67b substitution was sufficient, by itself, to lead to unprotection of the ester bond. Therefore, our finding strongly suggests that SePF is unable to interact with a tRNA(Sec) mutant version carrying a Watson-Crick G5a-C67b instead of the wild-type G5a x U67b base pair, establishing that G5a x U67b constitutes a structural determinant for SePF interaction.
authors.affiliation: Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan. mizutani@phar.nagoya-cu.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Mizutani
authors.forename: K
authors.initials: K
authors.lastname: Tanabe
authors.forename: K
authors.initials: K
authors.lastname: Yamada
mesh_terms: D000818:Animals; D001482:Base Composition; D002417:Cattle; D006147:Guanine; D008862:Microsomes, Liver / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010445:Peptide Elongation Factors / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017279:Selenocysteine / Q000378:metabolism*; D014498:Uracil
chemical_list: D010445:Peptide Elongation Factors; D012345:RNA, Transfer, Amino Acid-Specific; C036928:tRNA, selenocysteine-; D017279:Selenocysteine; D014498:Uracil; D006147:Guanine