title: Mutational identification of an essential tryptophan in tryptophanyl-tRNA synthetase of Bacillus subtilis.
abstract: The strongly conserved single tryptophan residue, Trp92, in Bacillus subtilis tryptophanyl-tRNA synthetase has been mutagenized via site direction singly into Gln, Ala, and Phe. All three mutant enzymes were inactive toward the catalysis of tRNA tryptophanylation. The Trp92----Phe mutant has been subcloned into the high expression plasmid pKK223-3 to yield the recombinant plasmid pKSW-F92. Growth of bacteria carrying the latter plasmid made possible the purification of the mutant TrpRS-F92 enzyme to homogeneity. This mutant enzyme was deficient in ultraviolet absorbance and fluorescence relative to the wild type enzyme and inactive in the partial reaction of Trp-activation as well as the overall reaction of tRNA tryptophanylation. Furthermore, unlike the wild type B. subtilis trpS gene, the mutant trpS-F92 gene upon transformation into Escherichia coli trpS 10343 failed to complement the temperature sensitive trpS mutation of the host cells. Trp92 therefore represents an essential residue both in vitro and in vivo for the function of the tryptophanyl-tRNA synthetase.
authors.affiliation: Department of Biochemistry, University of Toronto, Canada.
authors.forename: K C
authors.initials: KC
authors.lastname: Chow
authors.forename: H
authors.initials: H
authors.lastname: Xue
authors.forename: W
authors.initials: W
authors.lastname: Shi
authors.forename: J T
authors.initials: JT
authors.lastname: Wong
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*; D014369:Tryptophan-tRNA Ligase / Q000235:genetics*
chemical_list: D014364:Tryptophan; D014369:Tryptophan-tRNA Ligase