title: Dominant negative inhibition by fragments of a monomeric enzyme.
abstract: Dominant negative inhibition is most commonly seen when a mutant subunit of a multisubunit protein is coexpressed with the wild-type protein so that assembly of a functional oligomer is impaired. By analogy, it should be possible to interfere with the functional assembly of a monomeric enzyme by interfering with the folding pathway. Experiments in vitro by others suggested that fragments of a monomeric enzyme might be exploited for this purpose. We report here dominant negative inhibition of bacterial cell growth by expression of fragments of a tRNA synthetase. Inhibition is fragment-specific, as not all fragments cause inhibition. An inhibitory fragment characterized in more detail forms a specific complex with the intact enzyme in vivo, leading to enzyme inactivation. This fragment also associated stoichiometrically with the full-length enzyme in vitro after denaturation and refolding, and the resulting complex was catalytically inactive. Inhibition therefore appears to arise from an interruption in the folding pathway of the wild-type enzyme, thus suggesting a new strategy to design dominant negative inhibitors of monomeric enzymes.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Michaels
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
authors.forename: K
authors.initials: K
authors.lastname: Shiba
authors.forename: W T
authors.initials: WT
authors.lastname: Miller
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis / Q000235:genetics*; D004794:Enzyme Repression / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D009154:Mutation; D017510:Protein Folding
chemical_list: D000604:Amino Acyl-tRNA Synthetases