The initiation of transcription is one of the primary points for the regulation of gene expression. Specific protein-DNA and protein-protein interactions determine which genes are expressed in both normal and abnormal conditions. These interactions between macromolecules are determined by their three dimensional structures. Consequently, a molecular understanding of the protein-DNA and protein-protein interactions that are the core of a fundamental process like the regulation of gene expression requires a knowledge of the three-dimensional structures of the macromolecules that are involved. The proposed research will utilize X-ray crystallographic techniques to determine the three-dimensional structures of complexes of the bacteriophage lambda Cro repressor with a variety of oligonucleotides. These structural studies will generate detailed information on the protein- DNA interactions that can occur when proteins like Cro bind DNA in a base sequence independent fashion or to specific base sequences. Because it will be many years before the structures of all the proteins involved in the regulation of gene expression are known, model systems must be chosen that will provide general information. The Cro repressor provides an excellent system for studying the molecular basis for protein-DNA interactions. A second long term objective of the research is to examine how transcriptional regulatory proteins that recognize specific base sequences of DNA activate or repress the action of RNA polymerase. To affect transcription of a specific gene, the information that regulatory proteins are bound to particular base sequences must be transmitted to RNA polymerase. The enzyme responsible for the production of mRNA in eucaryotes, RNA polymerase II, contains an unusual repeating amino acid sequence will be examined using fluorescence energy transfer measurements and computer modelling. This structural information can then be related to the functional role of this portion of RNA polymerase II in the initiation of transcription and the regulation of gene expression.
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