Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.

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