title: Recognition of acceptor-stem structure of tRNA(Asp) by Escherichia coli aspartyl-tRNA synthetase.
abstract: Protein-RNA recognition between aminoacyl-tRNA synthetases and tRNA is highly specific and essential for cell viability. We investigated the structure-function relationships involved in the interaction of the Escherichia coli tRNA(Asp) acceptor stem with aspartyl-tRNA synthetase. The goal was to isolate functionally active mutants and interpret them in terms of the crystal structure of the synthetase-tRNA(Asp) complex. Mutants were derived from Saccharomyces cerevisiae tRNA(Asp), which is inactive with E. coli aspartyl-tRNA synthetase, allowing a genetic selection of active tRNAs in a tRNA(Asp) knockout strain of E. coli. The mutants were obtained by directed mutagenesis or library selections that targeted the acceptor stem of the yeast tRNA(Asp) gene. The mutants provide a rich source of tRNA(Asp) sequences, which show that the sequence of the acceptor stem can be extensively altered while allowing the tRNA to retain substantial aminoacylation and cell-growth functions. The predominance of tRNA backbone-mediated interactions observed between the synthetase and the acceptor stem of the tRNA in the crystal and the mutability of the acceptor stem suggest that many of the corresponding wild-type bases are replaceable by alternative sequences, so long as they preserve the initial backbone structure of the tRNA. Backbone interactions emerge as an important functional component of the tRNA-synthetase interaction.
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison, Madison, Wisconsin 53706-1567, USA.
authors.forename: Hyunsic
authors.initials: H
authors.lastname: Choi
authors.forename: Kay
authors.initials: K
authors.lastname: Gabriel
authors.forename: Jay
authors.initials: J
authors.lastname: Schneider
authors.forename: Sharee
authors.initials: S
authors.lastname: Otten
authors.forename: William H
authors.initials: WH
authors.lastname: McClain
mesh_terms: D001226:Aspartate-tRNA Ligase / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D011485:Protein Binding / Q000502:physiology; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D012349:RNA, Transfer, Asp; D001226:Aspartate-tRNA Ligase