Protein DNA Interactions Probed by UV Resonance Raman Spectroscopy

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9507241 Mukerji This research addresses molecular recognition and specificity in genetic regulatory processes which are mediated by protein binding, using a combination of spectroscopic and biochemical approaches. An understanding of these processes is gained by directly studying the molecular contacts defining the protein-nucleotide interface, using UV resonance Raman (UVRR) spectroscopy. This spectroscopic technique investigates the sequence specificity of the protein-DNA interaction by monitoring the H-bond pairings of nucleotide exocyclic amino and carbonyl groups. Additionally, the Raman effect is enhanced by tuning into an absorption band; therefore, different regions of the binary complex can be probed by varying the excitation wavelength. Thus, in addition to identifying the contacts which confer specificity to the binding site, UVRR spectroscopy also monitors conformational changes in both the protein and DNA. Investigations are conducted on two proteins, HU and Integration Host Factor(IHF), both members of a class of prokaryotic histone- like proteins. These proteins facilitate the binding and action of regulatory proteins by binding to the minor groove and bending the DNA. The majority of previously characterized protein-nucleic acid interactions have typically involved direct contact between the protein -helix and the major groove. In HU and IHF, binding and recognition occurs between -strands and the minor groove, representative of a different class of interaction. To date very few crystal structures exist for this type of protein-DNA interaction and this research provides a mechanism for identifying the factors which confer specificity and stability to the nucleosome complex. %%% Understanding the growth and development of cells centers on an understanding of the factors that regulate growth. At the most basic level this involves knowing how genes work and the factors that cause them to "turn on or turn off." In this research the mec hanism of regulation is studied by examining the interface between genetic regulatory proteins and the gene or the DNA. These proteins bind to the gene and either promote or repress its function. To understand how these proteins function, it is important to know in detail how they interact with DNA. A laser spectroscopic technique, UV resonance Raman spectroscopy, is used to investigate the interface between the proteins and the DNA. This method monitors the internal vibrations of molecules, which are a sensitive measure of the structure and bonding of a molecule. Using this technique, different parts of the protein or the DNA can be selectively examined, thereby allowing direct investigation of the important regions and contact points. These experiments probe the complex formed between regulatory proteins and genes and contribute towards understanding how genetic function is regulated in the cell. ***