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The RITE assay: identifying effectors that target the transcription machinery using phage display technology
J M Mazzarelli1, R P Ricciardi
1University of Pennsylvania, Philadelphia, PA, USA.
Abstract:
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.
Insights
Researchers developed a new assay to find transcription activators and repressors. This method, the reverse identification of transcriptional effectors (RITE) assay, directly identifies proteins that interact with transcription machinery.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Transcription regulation involves complex interactions between general transcription factors and various effector proteins.
- Identifying these effectors is crucial for understanding gene expression control.
- Existing methods like the yeast two-hybrid system have limitations in identifying transcription factor interactions.
Purpose of the Study:
- To introduce a novel assay, the reverse identification of transcriptional effectors (RITE) assay, for identifying transcription activators and repressors.
- To demonstrate the utility of phage display for discovering novel protein-protein interactions within the transcription machinery.
- To provide a method that directly measures interactions with specific transcription factor components.
Main Methods:
- Utilizing phage display to screen a library of cDNA-encoded peptides against specific regions of general transcription factors.
- Determining the amino acid sequence of interacting phage-displayed peptides.
- Performing database homology searches to identify potential effector proteins.
- Synthesizing candidate effectors and validating their binding and functional activity in vivo.
Main Results:
- The RITE assay successfully identifies proteins that interact with targeted components of the general transcription machinery.
- Homology searches enable the identification of known or novel transcription factors.
- Validated candidate effectors demonstrate binding to target proteins and modulate transcription in vivo.
Conclusions:
- The RITE assay is an effective method for identifying transcription effectors by directly targeting transcription machinery components.
- This approach offers advantages over existing methods like the yeast two-hybrid system for studying transcription factor interactions.
- The RITE assay facilitates the discovery of novel regulators of transcription.
