Evolutionary coadaptation of the motif 2 - Acceptor stem interaction in the class II prolyl-tRNA synthetase system

Known crystal structures of class II aminoacyl-tRNA synthetases complexed t o their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta -strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essenti al for tRNA aminoacylation but played no role in amino acid activation. Cro ss-linking experiments confirmed that the end of the tRNA(Pro) acceptor ste m is proximal to this motif 2 loop residue. Previous work had shown that th e tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved amo ng bacteria) are important recognition elements for E. coli ProRS. We carri ed out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G7 2 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chi meric tRNA(Pro) constructs showed that, in addition to A73 and G72, transpl antation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In co ntrast to the bacterial system, base-specific acceptor stem recognition doe s not appear to be used by human ProRS. Alanine-scanning mutagenesis reveal ed that motif 2 loop residues are not critical for tRNA aminoacylation acti vity of the human enzyme. Taken together, our results illustrate how synthe tases and tRNAs have coadapted to changes in protein-acceptor stem recognit ion through evolution.