Polynucleotide High Performance Affinity Chromatography

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DESCRIPTION (provided by applicant): The goal is to improve the performance DNA-affinity chromatography of transcription factors and to increase understanding of genetic regulation. To accomplish this we will: 1. Develop protocols we call "rational trapping" to allow oligonucleotide trapping to be easily optimized for a particular protein. Previously, we had developed a method called "oligonucleotide trapping" in which a probe DNA is mixed with cell extract at very low concentrations and with various competitors which lessen nonspecific binding. The protein-DNA complex is then rapidly isolated by chromatography and the transcription factor elutes in a high state of purity, typically homogeneous. The current method takes a long time to refine. We will use electrophoretic mobility shift assays to develop a rigorous, rational way to arrive at the correct conditions for successful purification. 2. The application of rational trapping to three very different transcription factors will show if it functions well for diverse proteins. The method will then be applied to three other transcription factors to show that it works well for diverse proteins from diverse organsims. 3. A new method, called promoter trapping, will be developed. The c-jun promoter is only about 200 bp. We will extend this trapping concept to purify the entire transcription pre-initiation complex (PIC) and characterize its component proteins by a proteomics approach using trypsin digestion and the mass spectrometers. This technique will allow identification of all components of the PIC, including ones which are currently unknown. The method, along with rational trapping, will then be used to purify three of the transcription factors bound by this promoter to investigate transcriptional regulation by cell signaling/phosphorylation in HeLa cells in response to serum mitogens. One of these transcription factors is currently unknown and will be characterized here for the first time.