Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA(Gln) interaction.

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.