Dominant Negative Inhibition by Fragments of a Monomeric Enzyme

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 25; pp. 14452 - 14455
• Main Authors: Michaels, John-Edward A., Schimmel, Paul, Shiba, Kiyotaka, Miller, W. Todd
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 10.12.1996; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences of the USA
• Subjects: Amino Acyl-tRNA Synthetases - biosynthesis; Amino Acyl-tRNA Synthetases - genetics; Antibodies; Bacteria; Biochemistry; Biological Sciences; Cell growth; Cellular biology; Elution; Enzyme Repression - genetics; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - growth & development; Gels; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Mutation; Plasmids; Protein Folding; Protein refolding; Proteins; Ribonucleic acid; RNA; Transfer RNA
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.25.14452

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.