Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accuratelyjoining 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 adenylateanalogue 5'-
O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (
KD)for cognate tRNA
His by more than 3-fold (from 3.87 to 1.17
M), and doubled the apparent
KD fornoncognate tRNA
Phe (from 7.3 to 14.5
M). By contrast, no binding discrimination against mutant U73tRNA
His was observed, even in the presence of HSA. Additional filter binding studies showed tighterbinding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., andFrancklyn, C. (1996)
Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminalanticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentationvelocity experiments, which showed that a stable complex was formed with the cognate tRNA
His but notwith noncognate tRNA
Phe. Footprinting experiments on wild-type versus G405D HisRS revealedcharacteristic 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 anticodonand core regions play major roles in the initial binding discrimination between cognate and noncognatetRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at stepsafter binding of tRNA.
