The goal of this application is an increased understanding of gene regulation. This subject will be approached from two different viewpoints--mechanisms controlling molecular interactions among regulatory molecules will be analyzed, and the overall behavior of a complex gene regulatory network will be explored. These issues will be studied using a combination of genetics and biochemistry, using two bacteriophages: lambda, the best-understood example of gene regulatory circuitry, and the lambda-related HK022. At the mechanistic level, the work will focus on a type of DNA binding termed cooperative binding. When one binding site is occupied by a protein, the affinity of a protein for an adjacent site is increased. Cooperative binding is involved in diverse DNA-related processes, including DNA replication, recombination, and initiation of transcription, and generally involves protein-protein contacts between DNA-bound proteins. It will be studied in the CI repressor of phage HK022. Mutant proteins defective in cooperativity will be studied, both to determine how the mutations interfere with cooperativity and to test whether cooperativity involves conformational changes in the protein. These studies are of general interest because they should shed light on cooperative interactions in complex systems with multiple components. At the level of gene regulatory circuitry, it will be asked how gene regulatory circuits behave, using the """"""""genetic switch"""""""" of phage. Lambda can exist in either of two alternative regulatory states. A """"""""minimal switch"""""""" will be developed to study the lambda system and mutated versions that are impaired in their ability to exist in two states. This minimal switch will be used to explore the behavior of genetic switches. Two properties, stability and robustness, that are thought to be crucial elements of such switches will be examined. These studies are of general interest because complex patterns of gene regulation exist in all organisms. For instance, development and pattern formation in higher eukaryotes involve a progressive determination of cell fate that is largely controlled by complex gene regulatory circuits.
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