The double helix DNA fragment we are studying is only a small part of the huge molecule which regulates, in the cell nucleus, the synthesis of proteins. Our DNA undecamer contains the consensus sequence recognized by sigma-k RNA polymerase, located in the promoter region of various genes expressed during sporulation of Bacillus subtilis. This is a rather complicated process where different co-ordinately controlled sets of genes are switched on successively during the course of sporulation. The expression of one set causes activation of the following set in the cascade. RNA polymerase containing the sigma-k factor has been shown able to transcribe various of these genes in vitro. Studying the structure of this DNA undecamer as well as other DNA fragments that interact with proteins provides us with details of those sequence dependent three-dimensional features that are essential for DNA recognition by proteins. This information will allow us to have a better understanding of biological processes that involve DNA recognition, e.g. gene expression. I solved the three-dimensional detailed structure of the DNA undecamer by using experimental information extracted from two-dimensional NMR spectra. Namely, interproton distances and torsional angles were extracted from 2D-NOE and 2QF-COSY spectra, respectively. I used this experimental information to restrain the structure of a starting DNA model during simulation of molecular dynamics (restrained molecular dynamics, r-MD). Different starting geometries have shown to converge, after r-MD, to the same final structure. Convergence indicates that experimental data are consistent and that the structure obtained resembles the three-dimensional structure of the DNA undecamer in solution. Back-calculation of the spectra by using the refined structures reproduced the experimental ones. Visual inspection of the DNA models with the program MidasPlus, before, during and after simulations, is an essential part of the process of refining and analyzing the final NMR structure. CGL resources have also been used to generate DNA models used for dynamic simulations. AMBER is the computer program used to refine the three-dimensional structure of the DNA fragment by running restrained molecular dynamics. I use the graphics program MidasPlus, available on the Computer Graphics Laboratory machines, to analyze the structure of enzymes and other biologically interesting molecules. This has been essential to successfully completion of my research work.
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