PROTEIN FOOTPRINTING OF RNASE PEPTIDE

We are continuing to explore the densitometric analysis of [unreadable]OH footprint autoradiograms. One approach that was explored during the past year is a [unreadable]band-by-band[unreadable] fitting analysis originally developed in our laboratory for thermodynamic studies using [unreadable]OH and 5-phenyl-phenanthroline as probes of DNA conformation that occur in sequence-specific protein-DNA interactions. Implementation of these protocols have been greatly facilitated by the development by Dr. Thomas Tullius and his co-workers of a band-fitting program (GelExplorer) with an interactive graphical interface interactive that runs within the IRIS Explorer environment. The results obtained by this procedure for the DNA containing a [unreadable]TATA Box[unreadable] (the binding site of the eukaryotic transcription factor TBP) shows that the decrease in Hthe relative band densities for the sequence [unreadable]AAAA[unreadable] is consistent with Hthe assumption of an altered configuration by this sequence. Similar Hdata sets obtained at temperatures from 15 - 45 ?C are being compared Hwith solvent accessibility calculations of this DNA obtained from Hmolecular dynamics calculations. The goal of this project is to correlate changes in DNA conformation with the temperature dependencies of the thermodynamics and kinetics of DNA binding by TBP. H[unreadable]OH cleavage data sets have been obtained for a number of different H[unreadable]TATA box[unreadable] sequences that display unique thermodynamic and kinetic Hsignatures.