title: Aptamer redesigned tRNA is nonfunctional and degraded in cells.
abstract: An RNA aptamer derived from tRNA(Gln) isolated in vitro and a rationally redesigned tRNA(Gln) were used to address the relationship between structure and function of tRNA(Gln) aminoacylation in Escherichia coli. Two mutant tRNA(Gln) sequences were studied: an aptamer that binds 26-fold tighter to glutaminyl-tRNA synthetase than wild-type tRNA(Gln) in vitro, redesigned in the variable loop, and a mutant with near-normal aminoacylation kinetics for glutamine, redesigned to contain a long variable arm. Both mutants were tested in a tRNA(Gln) knockout strain of E. coli, but neither supported knockout cell growth. It was later found that both mutant tRNAs were present in very low amounts in the cell. These results reveal the difference between in vitro and in vivo studies, demonstrating the complexities of in vivo systems that have not been replicated in vitro.
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706-1569, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Lee
authors.forename: William H
authors.initials: WH
authors.lastname: McClain
mesh_terms: D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids; D012329:RNA, Bacterial / Q000737:chemistry*; D012351:RNA, Transfer, Gln / Q000235:genetics* / Q000378:metabolism*
chemical_list: D012329:RNA, Bacterial; D012351:RNA, Transfer, Gln; D005973:Glutamine; D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase