The RITE assay: identifying effectors that target the transcription machinery using phage display technology.
We describe an approach using phage display to identify effectors (activators and repressors) of transcription based on the particular component of the general transcription machinery that they target. We refer to this approach as the reverse identification of transcriptional effectors (RITE) assay. A library of phages containing cDNA-encoded peptides displayed on their surfaces is screened using as the target a specific region of one of the general transcription factors (e.g., the C terminus of hTAFII135). The amino acid sequence encoded by the cDNA of an interacting phage is determined and analyzed in a database homology search to identify known or novel factors that may interact with the target protein. Candidate effectors from the homology search are synthesized from recombinant clones and tested for their abilities to bind to the target protein and to functionally modulate transcription in vivo when co-expressed with the transcriptional target protein. Because the RITE assay is a direct measure of the interactions between general transcription proteins and their effectors, it has an advantage over the well-known yeast two-hybrid system, which is not amenable to identifying transcription factor interactions.
University of Pennsylvania, Philadelphia, PA, USA.
J M
JM
Mazzarelli
R P
RP
Ricciardi
D000595:Amino Acid Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000502:physiology; D000072482:Retinoic Acid Receptor alpha; D014158:Transcription, Genetic / Q000187:drug effects*
D022763:CCAAT-Enhancer-Binding Protein-alpha; D019151:Peptide Library; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha