Mimics of yeast tRNA(Asp) and their recognition by aspartyl-tRNA synthetase

Assuming that the L-shaped three-dimensional structure of tRNA is an archit ectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA ar chitectures can fulfill this role and be functional substrates of these enz ymes, provided they contain correctly located identity elements. In this wo rk, this paradigm was submitted to new experimental verification. Yeast asp artyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNA(Asp) can be altered without losing its ability to be aminoacylated was investigated. Three novel archi tectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNA(Ser) lacking the D-arm, The second lacks bot h the D- and T-arms, and the 5'-strand of the amino acid acceptor arm. The third structure is a construct in which the acceptor and anticodon helices are joined by two connectors. Aspartylation specificity of these RNAs is ve rified by the loss of aminoacylation activity upon mutation of the putative identity residues. Kinetic data indicate that the first two architectures are mimics of the whole rRNA(Asp) molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the di scriminator nucleotide G73 in aspartylation and demonstrate that neither a helical structure in the acceptor domain nor the presence of a D- or T-arm is mandatory for specific aspartylation, but that activity relies on the pr esence of the cognate aspartate GUC sequence in the anticodon loop.