title: Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction.
abstract: This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA(Gln). Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA(Gln).GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA(Gln) species were synthesized with either a 2'-deoxy AMP or 3'-deoxy AMP as their 3'-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PPi exchange activity established the esterification of glutamine to the 2'-hydroxyl of the terminal adenosine; there is no glutaminylation of the 3'-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA(Gln) are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
authors.forename: S
authors.initials: S
authors.lastname: Englisch-Peters
authors.forename: J
authors.initials: J
authors.lastname: Conley
authors.forename: J
authors.initials: J
authors.lastname: Plumbridge
authors.forename: C
authors.initials: C
authors.lastname: Leptak
authors.forename: D
authors.initials: D
authors.lastname: Söll
authors.forename: M J
authors.initials: MJ
authors.lastname: Rogers
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005975:Glutamate-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D012329:RNA, Bacterial; D012351:RNA, Transfer, Gln; D005975:Glutamate-tRNA Ligase