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

Abstract

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 tRNAHis, 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 tRNAHis, 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, microhelixHis is a much poorer substrate relative to full-length tRNAHis for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5′ monophosphate of yeast tRNAHis 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 tRNAHis appears to be to properly position the critical 5′ monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNAHis interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system. Copyright © 2006 RNA Society.