title: Mimics of yeast tRNAAsp and their recognition by aspartyl-tRNA synthetase.
abstract: Assuming that the L-shaped three-dimensional structure of tRNA is an architectural framework allowing the proper presentation of identity nucleotides to aminoacyl-tRNA synthetases implies that altered and/or simplified RNA architectures can fulfill this role and be functional substrates of these enzymes, provided they contain correctly located identity elements. In this work, this paradigm was submitted to new experimental verification. Yeast aspartyl-tRNA synthetase was the model synthetase, and the extent to which the canonical structural framework of cognate tRNAAsp can be altered without losing its ability to be aminoacylated was investigated. Three novel architectures recognized by the synthetase were found. The first resembles that of metazoan mitochondrial tRNASer lacking the D-arm. The second lacks both 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 verified 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 tRNAAsp molecule, while the third one behaves as an aspartate minihelix mimic. Results confirm the primordial role of the discriminator 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 presence of the cognate aspartate GUC sequence in the anticodon loop.
authors.affiliation: UPR "Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance", Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
authors.forename: A D
authors.initials: AD
authors.lastname: Wolfson
authors.forename: A M
authors.initials: AM
authors.lastname: Khvorova
authors.forename: C
authors.initials: C
authors.lastname: Sauter
authors.forename: C
authors.initials: C
authors.lastname: Florentz
authors.forename: R
authors.initials: R
authors.lastname: Giegé
mesh_terms: D000215:Acylation; D001226:Aspartate-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000235:genetics; D005818:Genetic Engineering; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000138:chemical synthesis; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae
chemical_list: D012349:RNA, Transfer, Asp; D001226:Aspartate-tRNA Ligase