INTERACTIONS BETWEEN TRANSFER-RNA IDENTITY NUCLEOTIDES AND THEIR RECOGNITION SITES IN GLUTAMINYL-TRANSFER-RNA SYNTHETASE DETERMINE THE COGNATE AMINO-ACID AFFINITY OF THE ENZYME

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent t he binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme mo dulate the amino acid affinity of GlnRS. Perturbation of any of the pr otein-RNA interactions through mutation of either component led to con siderable changes in glutamine affinity with the most marked effects s een at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based up on biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act a s strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair acid U35 are apparently t he major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amin o acid recognition reveals a novel mechanism for maintaining translati onal fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.