title: Site-directed mutagenesis to fine-tune enzyme specificity.
abstract: We have used a combination of a genetic selection and oligonucleotide-directed mutagenesis to introduce a series of amino acid replacements for a single residue into Escherichia coli glutaminyl-tRNA synthetase. The mutant enzymes mischarge supF tRNA(Tyr), with glutamine, to varying degrees depending on the polarity of the side chain introduced but apparently not depending on the size or shape of the side chain. These results indicate that repulsive charge-charge interactions may be important for specific recognition of nucleic acids by proteins and illustrate how a mutant, derived from genetic selection, may be further modified in activity by oligonucleotide-directed mutagenesis.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
authors.forename: H
authors.initials: H
authors.lastname: Uemura
authors.forename: M J
authors.initials: MJ
authors.lastname: Rogers
authors.forename: R
authors.initials: R
authors.lastname: Swanson
authors.forename: L
authors.initials: L
authors.lastname: Watson
authors.forename: D
authors.initials: D
authors.lastname: Söll
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004274:DNA, Recombinant / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods; D005975:Glutamate-tRNA Ligase / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D012641:Selection, Genetic; D013379:Substrate Specificity; D013489:Suppression, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D004274:DNA, Recombinant; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases; D005975:Glutamate-tRNA Ligase