INVITRO SYNTHESIS OF SELENOCYSTEINYL-TRANSFER RNAUCA FROM SERYL-TRANSFER RNAUCA - 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 ester-bonded to tRNAUCASer- tRNAUCASer 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-tRNAUCASer 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 tRNAUCA and they are in accord with SelD functioning as a donor of reduced selenium.