A General Strategy to Enhance the Potency of Chimeric Transcriptional Activators

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 96; no. 24; pp. 13898 - 13903
• Main Authors: Natesan, Sridaran, Molinari, Elizabeth, Rivera, Victor M., Rickles, Richard J., Gilman, Michael
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23.11.1999; National Acad Sciences; National Academy of Sciences
• Subjects: Amino acids; Binding Sites; Biological Sciences; Bundling; Cell lines; Cells; Cloning, Molecular; Gene expression; Gene therapy; Genes; Genetics; Herpes Simplex Virus Protein Vmw65 - genetics; Humans; Immunophilins - genetics; NF-kappa B - genetics; Plasmids; Proteins; Recombinant Fusion Proteins - genetics; Reporter genes; Tacrolimus Binding Proteins; Trans-Activators - metabolism; Transcription factors; Tumor Cells, Cultured; Two hybrid system techniques
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.24.13898

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.