X-RAY DIFFRACTION ANALYSIS OF DNA BINDING PROTEINS

点击翻译按钮获取中文摘要

This research will establish precisely defined protein/nucleic acid structural systems though the application of X-ray crystallographic techniques for the purpose of providing a sound physical basis for the description of protein-nucleic acid interactions. This will assist molecular biologists in deducing, at an atomic level, the means by which genetic information is regulated and transformed into specific cellular events. It furthermore will contribute to our understanding of the fundamental principles underlying the structure of the chromosome. By direct visualization of the crucial interactions, the structural elements of these macromolecules responsible for their biological activity can be explicitly defined. This research will encompass the three-dimensional structure solution and high precision refinement of the Gene 5 protein from fd bacteriophage and deoxyribonuclease from beef pancreas. In addition, complexes of these proteins with oligo-deoxynucleotides will be analyzed by difference Fourier and molecular replacement techniques to map the crucial atomic interactions. Mutant forms of the Gene 5 protein and homologous proteins from closely related viruses will be analyzed by crystallographic analysis to derive those molecular details particularly sensitive to modification Attempts will also be made to isolate, purify and crystallize DNA binding proteins from other organisms for the purpose of expanding the range of structures and interactions available for single crystal X-ray diffraction analyses. The initial efforts in this regard will be directed toward the DNA unwinding proteins from T4 bacteriophage (the Gene 32 protein), E. coli and calf thymus, the DNA binding protein from cultured human fibroblasts and the lactose operon repressor from E. coli.