In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-01, Vol.87 (2), p.543-547
• Main Authors: Leinfelder, W, Forchhammer, K, Veprek, B, Zehelein, E, Böck, A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Base Sequence; Codon - genetics; Drosophila Proteins; Escherichia coli - genetics; Genes, Bacterial; Genotype; Molecular Sequence Data; Mutation; Phosphotransferases; Plasmids; Restriction Mapping; RNA, Transfer, Amino Acid-Specific - metabolism; RNA, Transfer, Amino Acyl - biosynthesis; RNA, Transfer, Amino Acyl - genetics; RNA, Transfer, Ser - metabolism; Selenium - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.2.543

The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue esterbonded to tRNA(Ser)(UCA). tRNA(Ser)(UCA) has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNA(Ser)(UCA) was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNA(UCA) and they are in accord with SelD functioning as a donor of reduced selenium.