Conservation in Evolution for a Small Monomeric Phenylalanyl-tRNA Synthetase of the tRNAPhe Recognition Nucleotides and Initial Aminoacylation Site

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Abstract
We previously showed that yeast mitochondrial phenylalanyl-tRNA synthetase (MSF protein) is evolutionarily distant to the cytoplasmic counterpart based on a high degree of divergence in protein sequence, molecular mass, and quaternary structure. Using yeast cytoplasmic tRNAPhe which is efficiently aminoacylated by MSF protein, we report here the tRNAPhe primary site of aminoacylation and the identity determinants for MSF protein. As for the cytoplasmic phenylalanyl-tRNA synthetase (Sampson, J. R., Di Renzo, A. B., Behlen, L. S., & Uhlenbeck, O. C. (1989) Science 243, 1363−1366), MSF protein recognizes nucleotides from the anticodon and the acceptor end including base A73 and, as shown here, adjacent G1-C72 base pair or at least C72 base. This indicates that the way of tRNAPhe binding for the two phenylalanine enzymes is conserved in evolution. However, tRNAPhe tertiary structure seems more critical for the interaction with the cytoplasmic enzyme than with MSF protein, and unlike cytoplasmic phenylalanyl-tRNA synthetase, the small size of the monomeric MSF protein probably does not allow contacts with residue 20 at the top corner of the L molecule. We also show that MSF protein preferentially aminoacylates the terminal 2‘-OH group of tRNAPhe but with a catalytic efficiency for tRNAPhe-CC-3‘-deoxyadenosine reduced 100-fold from that of native tRNAPhe, suggesting a role of the terminal 3‘-OH in catalysis. The loss is only 1.5-fold when tRNAPhe-CC-3‘-deoxyadenosine is aminoacylated by yeast cytoplasmic PheRS (Sprinzl, M., & Cramer, F. (1973) Nature 245, 3−5), indicating mechanistic differences between the two PheRS's active sites for the amino acid transfer step.