GENOMIC ORGANIZATION, CDNA SEQUENCE, BACTERIAL EXPRESSION, AND PURIFICATION OF HUMAN SERYL-TRANSFER-RNA SYNTHASE

In this paper, we report the cDNA sequence and deduced primary sequenc e for human cytosolic seryl-tRNA synthetase, and its expression in Esc herichia coli. Two human brain cDNA clones of different origin, contai ning overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone) . For both clones the 5' region of the cDNA was missing. This 5' regio n was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to is olate and characterize the intron/exon structure of the serS gene, con sisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp . The availability of the gene structure of the human enzyme could hel p to clarify some aspects of the molecular evolution of class-II amino acyl-tRNA synthetases. The human seryl-tRNA synthetase has been expres sed in E. coli, purified (95% pure as determined by SDS/PAGE) and kine tic parameters have been measured for its substrate tRNA. The human se ryl-tRNA synthetase sequence (514 amino acid residues) shows significa nt sequence identity with seryl-tRNA synthetases from E. coli (25%), S accharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorha bditis elegans (60%). The partial sequences from published mammalian s eryl-tRNA synthetases are very similar to the human enzyme (94% and 92 % identity for mouse and Chinese hamster seryl-tRNA synthetase, respec tively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal do main and a N-terminal insertion identified in the human enzyme. In fun ctional studies, the enzyme aminoacylates calf liver tRNA and prokaryo tic E. coli tRNA.