Covariation of a specificity-determining structural motif in an aminoacyl-tRNA synthetase and a tRNA identity element

Transfer RNA (tRNA) identity determinants help preserve the specificity of aminoacylation in vivo, and prevent cross-species interactions. Here, we in vestigate covariation between the discriminator base (N73) element in histi dine tRNAs and residues in the histidyl-tRNA synthetase (HisRS) motif 2 loo p. A model of the Escherichia coli HisRS-tRNA(His) complex predicts an inte raction between the prokaryotic conserved glutamine 118 of the motif 2 loop and cytosine 73. The substitution of Gin 118 in motif 2 with glutamate dec reased discrimination between cytosine and uracil some 50-fold, but left ov erall rates of adenylation and aminoacylation unaffected. By contrast, subs titutions at neighboring Glu 115 and Arg 121 affected both adenylation and aminoacylation, consistent with their predicted involvement in both half-re actions. Additional evidence for the involvement of the motif 2 loop was pr ovided by functional analysis of a hybrid Saccharomyces cerevisiae- E. coli HisRS possessing the 11 amino acid motif 2 loop of the yeast enzyme. Despi te an overall decreased activity of nearly 1000-fold relative to the E. col i enzyme, the chimera nevertheless exhibited a modest preference for the ye ast tRNA(His) over the E. coli tRNA, and preferred wild-type yeast tRNA(His ) to a variant with C at the discriminator position. These experiments sugg est that part of, but not all of, the specificity is provided by the motif 2 loop. The close interaction between enzyme loop and RNA sequence elements suggested by these experiments reflects a covariation between enzyme and t RNA that may have acted to preserve aminoacylation fidelity over evolutiona ry time.