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Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs

¹ Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA
² Department of Biochemistry, University of Vermont, Health Sciences Complex, Burlington, Vermont 05405, USA

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5' 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 5'-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 –1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelix^His 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 5' 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 –1:73 base pair of yeast tRNA^His appears to be to properly position the critical 5' 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.

Keywords: yeast histidine-tRNA synthetase; tRNA recognition; acceptor stem microhelix; extra base pair; phosphate recognition; atomic group mutagenesis

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