The central objectives for the next period of funding are: (1) to characterize the conformational changes induced in DNA by two DNA binding proteins, namely, the cro repressor of bacteriophage lambda and the histone-like HU protein of E. coli; (2) to further characterize the geometries of immobile, four-way DNA junctions; and (3) the characterization of the conformational properties of several common elements of non-helical structure found in RNA. The two protein-DNA interactions represent paradigms for the study of sequence-specific and non-specific interactions, respectively; such interactions will be studied from the standpoint of the alterations in helix axis direction which accompany complex formation. The study of four-way DNA junctions is designed to increase our understanding of the conformational properties of cruciforms as well as intermediates in site-specific and general recombination. With regard to the latter, we hope eventually to provide a mechanistic basis for the interactions of recombinases/resolvases with branched DNA structures. The study of intermediate-to-long-range RNA structure, the major emphasis of the proposed research, should provide a unique perspective on the overall three-dimensional structures of RNA molecules. We know of no other studies of the type proposed herein. Our methods of approach to all three objectives are similar: combined gel electrophoretic and birefringence decay studies will be used to define alterations in the directions of DNA or RNA helix axes, which arise from the structural element being studied (protein-DNA complex; junction; non-helical element). Gel electrophoresis provides qualitative information pertaining to axial distortions, as a consequence of reductions in electrophoretic mobility that accompany axial curvature. Birefringence decay measurements, when combined with computational analyses of hydrodynamic behavior, provide more quantitative information regarding axial bending.
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