Human tryptophanyl-tRNA synthetase can efficiently complement the Saccharoymces cerevisiae homologue, Wrs1P.
Aminoacyl-tRNA synthetases corresponding to each of the 20 amino acids are essential proteins for nearly all cells. The tryptophan-specific enzyme in the cytoplasm of Saccharomyces cerevisiae (ScWRS) is a 49-kDa protein encoded by the WRS1 gene and required for survival. The human enzyme (HsWRS) is a 54-kDa protein with 46% sequence identity to ScWRS. HsWRS has a kinase domain in the N-terminal region and a serine phosphorylation site near the C-terminus not present in the yeast enzyme. To determine if the gene encoding the human protein could functionally complement the WRS1 gene, HsWRS cDNA was cloned in the expression vectors pVT100U and pYES then transformed into a diploid yeast where one copy of WRS1 had been replaced with a G418(R) gene. Tetrads derived from these transformants were dissected and spores germinated on media selecting for the presence of the plasmids. Haploid colonies were then tested for resistance to G418. G418(R) cells were unable to grow in the presence of 5-fluoroorotic acid, indicating their dependence on the plasmids for viability. Polymerase chain reaction analysis of these cells confirmed the presence of the HsWRS gene and the absence of WRS1. Growth rates of cells expressing HsWRS are essentially identical to the parent. This demonstrates that the human enzyme can function in yeast and effectively replace the ScWRS protein in spite of the presence of two unique functions and a >50% overall difference in amino acid sequence.
Johnson
Jerry D
JD
Department of Molecular Biology, P.O. Box 3944, University of Wyoming, Laramie, WY 82071, USA. jdjohn@uwyo.edu
Spellman
Jennifer M
JM
White
Karen H
KH
Barr
Kristine K
KK
John
Theodore R
TR
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D016678:Genome; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D014170:Transformation, Genetic; D014369:Tryptophan-tRNA Ligase / Q000032:analysis / Q000235:genetics*
D014369:Tryptophan-tRNA Ligase