DISSECTION OF A CLASS-II TRANSFER-RNA SYNTHETASE - DETERMINANTS FOR MINIHELIX RECOGNITION ARE TIGHTLY ASSOCIATED WITH DOMAIN FOR AMINO-ACID ACTIVATION

The ten class II aminoacyl-tRNA synthetases are large homo- and hetero-oligomeric proteins that share three conserved sequence motifs. Within this class, Escherichia coli alanyl-tRNA synthetase is the only homotetramer and is comprised of subunits of 875 amino acids. The enzyme aminoacylates sequence-specific RNA oligonucleotides that recreate as few as four base pairs of the acceptor stem of tRNA(Ala). A monomeric 461 amino acid N-terminal fragment (461N) was previously shown to have full adenylate synthesis activity. However, fragment 461N has significant, but reduced, efficiency of charging of tRNA(Ala), when compared to native enzyme [Ho, C., Jasin, M., & Schimmel, P. (1985) Science 229, 389-393]. We show here that, in contrast, the fragment and the native enzyme aminoacylate a 12 base pair acceptor-TpsiC stem minihelix and a four base pair RNA tetraloop with the same efficiency. We also show that fragment 461N makes footprint contacts both on and outside the acceptor helix of bound tRNA(Ala). With one possible exception, the contacts observed with fragment 461N are identical to those seen with the native enzyme. In spite of contacts outside the acceptor helix, fragment 461N charges the native tRNA, minihelix, and tetraloop with similar efficiency. Thus, all minihelix contacts required for activation for charging are tightly associated with the adenylate synthesis domain and, at least in the fragment, are not influenced by additional RNA-protein contacts outside the minihelix domain. These findings and other considerations suggest that RNA interactions essential for aminoacylation may have developed as an integral part of the site for amino acid activation and are restricted to nucleotides near the amino acid attachment site.