Structure determination of the lactose repressors

The goal of this thesis was to determine the x-ray crystal structure of the Lactose Repressor and its complex with DNA and inducer. These three crystal structures provided a structural basis for the allosterism associated with the transcriptional regulation of the Lac Operon, which is the paradigm for gene regulation. The structure of the Lactose Repressor complexed to two 21-bp DNA suggested a model for transcriptional regulation through a DNA looping mechanism. This has potential implications for eukaryotic systems where DNA looping transcriptional mechanisms are more prevalent. The x-ray structure determination of the Lactose Repressor was complicated and several new methods were developed to address specific crystallographic computing problems using genetic algorithms. This thesis discusses how genetic algorithms can be used to (i) solve heavy atom positions for the isomorphous replacement method, (ii) search for a molecular replacement solution, and (iii) protein phasing. The application of these new solving techniques and the non-crystallographic symmetry averaging/refinement procedures for the uncomplexed, repressed, and induced forms of the Lactose Repressor will be discussed. The thesis concludes with structural comparisons