A TRANSFER-RNA IDENTITY SWITCH MEDIATED BY THE BINDING INTERACTION BETWEEN A TRANSFER-RNA ANTICODON AND THE ACCESSORY DOMAIN OF A CLASS-II AMINOACYL-TRANSFER-RNA SYNTHETASE

Identity elements in tRNAs and the intracellular balance of tRNAs allo w accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histi dyl-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 m ajor identity element for histidine acceptance, such that the substitu tion of C73 brings about mischarging by glycyl-, glutaminyl-, and leuc yl-tRNA synthetases. These identity conversions mediated by the G-1 . C73 base pair were exploited to isolate secondary site revertants in t he histidyl-tRNA synthetase from E. coli which restore histidine ident ity to a histidyl-tRNA suppressor carrying U73. The revertant substitu tions confer a 3-4-fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal dom ain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant rol e in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kin etic parameters determined also show a small degree of anticooperativi ty (Delta Delta G = -1.24 kcal/mol) between recognition of the discrim inator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these e ffects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring abou t a complete conversion of tRNA identity.