tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic inform ation by accurately joining amino acids to their cognate transfer RNAs. Her e, tRNA discrimination at the level of binding by Escherichia call histidyl -tRNA synthetase is addressed by filter binding, analytical ultracentrifuga tion, and iodine footprinting experiments. Competitive filter binding assay s show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfa moyl] adenosine, HSA, decreased the apparent dissociation constant (K-D) fo r cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 mu M), and doubl ed the apparent K-D for noncognate tRNA(Phe) (from 7.3 to 14.5 mu M). By co ntrast, no binding discrimination against mutant U73 tRNA(His) was observed , even in the presence of HSA. Additional filter binding studies showed tig hter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Ya n, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], whic h possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimen tation 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 alterat ions in the pattern of protection and enhancement of iodine cleavage at pho sphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together , these results suggest that the anticodon and core regions play major role s in the initial binding discrimination between cognate and noncognate tRNA s, whereas acceptor stem nucleotides, particularly at position 73, influenc e the reaction at steps after binding of tRNA.