CONSERVATION IN EVOLUTION FOR A SMALL MONOMERIC PHENYLALANYL-TRANSFER-RNA SYNTHETASE OF THE TRNA(PHE) RECOGNITION NUCLEOTIDES AND INITIAL AMINOACYLATION SITE

We previously showed that yeast mitochondrial phenylalanyl-tRNA synthe tase (MSF protein) is evolutionarily distant to the cytoplasmic counte rpart based on a high degree of divergence in protein sequence, molecu lar mass, and quaternary structure. Using yeast cytoplasmic tRNA(Phe) which is efficiently aminoacylated by MSF protein, we report here the tRNA(Phe) 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. (1 989) Science 243, 1363-1366), MSF protein recognizes nucleotides from the anticodon and the acceptor end including base A(73) and, as shown here, adjacent G(1)-C-72 base pair or at least C-72 base. This indicat es that the way of tRNA(Phe) binding for the two phenylalanine enzymes is conserved in evolution. However, tRNA(Phe) tertiary structure seem s more critical for the interaction with the cytoplasmic enzyme than w ith MSF protein, and unlike cytoplasmic phenylalanyl-tRNA synthetase, the small size of the monomeric MSF protein probably does not allow co ntacts with residue 20 at the top corner of the L molecule. We also sh ow that MSF protein preferentially aminoacylates the terminal 2'-OH gr oup of tRNA(Phe) but With a catalytic efficiency for tRNA(Phe)-CC-3'-d eoxyadenosine reduced 100-fold from that of native tRNA(Phe), suggesti ng a role of the terminal 3'-OH in catalysis. The loss is only 1.5-fol d when tRNA(Phe)-CC-3'-deoxyadenosine is aminoacylated by yeast cytopl asmic PheRS (Sprinzl, M., & Cramer, F. (1973) Nature 245, 3-5), indica ting mechanistic differences between the two PheRS's active sites for the amino acid transfer step.