In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.
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.
Lehrstuhl für Mikrobiologie, Universität München, Federal Republic of Germany.
W
W
Leinfelder
K
K
Forchhammer
B
B
Veprek
E
E
Zehelein
A
A
Böck
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D009154:Mutation; D010770:Phosphotransferases; D010957:Plasmids; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000096:biosynthesis* / Q000235:genetics; D012362:RNA, Transfer, Ser / Q000378:metabolism*; D015183:Restriction Mapping; D012643:Selenium / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D029721:Drosophila Proteins; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012362:RNA, Transfer, Ser; C112705:selenocysteinyl-tRNA; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D012643:Selenium