We study the behavior of gene regulatory circuitry, using a combination of genetics and biochemistry, in the following two model systems:
The SOS response of E. coli is controlled by proteolytic cleavage of LexA repressor. In the cell, another protein, RecA, acts as a "coprotease" to stimulate LexA self-cleavage. We believe that RecA stabilizes a reactive form of LexA. Initial evidence for this came from analysis of mutant proteins with increased rates of cleavage. Recent crystal structures of several mutant forms of LexA show conclusively that the protein can exist in two distinct conformations: In one form, the cleavage site lies in the active site, adjacent to the catalytic center; in the other form, the cleavage site is about 20 Å from the cleavage site. We propose that RecA stabilizes the former conformation.
The "genetic switch" of phage lambda allows two mutually exclusive patterns of gene expression. It involves the action of two regulatory proteins, CI and Cro. Our work analyzes the systems behavior of this gene regulatory circuitry, focusing on several features. First, how stable are the regulatory states in the face of perturbations in CI and Cro levels? Second, how robust is the genetic switch? Can we change its components and still have a bistable switch? We find that the switch can be altered substantially and yet still function normally. Importantly, this indicates that the switch is robust, and implies that a switch could evolve by finding a workable circuit, then by refining it for optimal behavior. Third, what are the roles of cooperative DNA binding and positive autoregulation by CI protein in the lambda circuitry? Our evidence suggests that these activities of CI are not essential for proper operation of the genetic switch. Finally, what causes the threshold behavior of the prophage induction process, and how is the set-point determined? This work reveals an interaction between the oL and oR regulatory regions, specifically involving the oL3 site. All these studies have general implications for the evolution of complex gene regulatory circuits.
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Atsumi, S., and J.W. Little JW. 2006. Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach. Proceedings of the National Academy of Sciences U.S.A. 103: 4558-4563.
Michalowski, C.B., and J.W. Little JW. 2005. Positive autoregulation of cI is a dispensable feature of the phage lambda gene regulatory circuitry. Journal of Bacteriology 187: 6430-6442.
Little, J.W. 2005 Threshold effects in gene regulation: when some is not enough. Proceedings of the National Academy of Sciences U.S.A. 102: 5310-5311.
Michalowski, C.B., M.D. Short, and J.W. Little. 2004. Sequence tolerance of the phage lambda PRM promoter: implications for evolution of gene regulatory circuitry. Journal of Bacteriology 186: 7988-7999.
Atsumi, S., and J.W. Little. 2004. Regulatory circuit design and evolution using phage lambda. Genes and Development 18: 2086-2094.
Luo, Y., R.A. Pfuetzner, S. Mosimann, M. Paetzel, E.A. Frey, M. Cherney, B. Kim, J.W. Little, and Natalie C.J. Strynadka. 2001. Crystal structure of LexA: A conformational switch for regulation of self-cleavage. Cell 106: 585-594.
Mustard, J.A., and J.W. Little. 2000. Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of recA. Journal of Bacteriology 182: 1659-1670.
