All histidine tRNA molecules have an extra nucleotide, G-1, at the 59 end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNA His , G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNA His identity. Specifically, the 59-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute ; ;4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNA His , were used to facilitate an atomic group ''mutagenesis'' study of the ÿ1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNA His for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 59 monophosphate of yeast tRNA His is critical for aminoacylation by yeast HisRS and contributes ; ;3 kcal/mol to transition state stability. The primary role of the unique ÿ1:73 base pair of yeast tRNA His appears to be to properly position the critical 59 monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNA His interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.

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