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John Little

Education and Appointments

• Ph.D. 1967, Stanford University
  

Research Interests

Research Summary

Behavior and evolution of gene regulatory circuits. Mechanisms of gene regulation. Biochemistry of the SOS response in E. coli.

We seek to understand the behavior of gene regulatory circuitry and to analyze molecular interactions controlling regulatory circuits at the mechanistic level. We use a combination of genetics and biochemistry to approach these problems in two systems: The regulatory circuitry of bacteriophage lambda, the best understood genetic switch; and the specific cleavage reaction that controls the SOS regulatory system.

Lambda regulatory circuitry: The "genetic switch" of phage lambda allows a choice between two patterns of gene expression. This switch involves the interplay between two regulatory proteins, CI and Cro, which bind to a complex regulatory region termed oR. These proteins stabilize two mutually exclusive patterns of gene expression. The regulatory circuitry that controls these two alternatives is understood in considerable detail. Moreover, one of the patterns of gene expression (the "lysogenic" state) can be switched to the other (the "lytic" state) by treatments that damage DNA and induce the SOS response. This "genetic switch" has threshold behavior--that is, it occurs above a threshold level of damage, but not below that threshold.

We are interested in studying three features of this circuit: First, how stable are its states? Can they be maintained after perturbations in the levels of the regulatory proteins? Second, how robust is the genetic switch? Can it tolerate changes in the behavior of its components and still operate as a bistable switch? Our recent evidence suggests that the switch can be altered substantially and yet can still function normally. This evidence indicates that the switch is robust. It also has important implications for evolution: we suggest that a switch could evolve by first finding a workable circuit, then by refining this circuit for optimal behavior. Third, the genetic switch has a set-point; how is the set-point determined, and why is the threshold so sharp?

The SOS regulatory system controls the response of E. coli to treatments that damage DNA or inhibit DNA replication. This system is controlled by two proteins: the LexA repressor, which normally represses a set of about 20 genes; and the RecA protein, which is activated by inducing treatments and promotes a specific cleavage of LexA repressor. Our work focuses on this cleavage reaction. Cleavage is an inherent property of the repressor protein, which autodigests at high pH by a mechanism like that of a serine protease; RecA stimulates autodigestion, perhaps by stabilizing a conformation of LexA that is competent for cleavage. We are studying mutant proteins with increased rates of cleavage; using these mutants, we can make cleavage work in a bimolecular reaction, so that we can treat this reaction as a standard enzymatic reaction. Current work focuses on determining the chemical mechanism of cleavage, and on the role of RecA in stimulating this reaction.

Selected Publications

Giese, K.C., Michalowski, C.B. and Little, J.W. (2008). RecA-Dependent cleavage of LexA dimers. J. Mol. Biol. 377: 148-161.

Babic, A.C. and Little, J.W. (2007). Cooperative DNA binding by CI repressor is dispensable in a phage λ variant. Proc. Natl. Acad. Sci. USA: 104: 17741-17746.

Degnan, P.H., Michalowski, C.B., Babic, A.C., Cordes, M.H.J. and Little, J.W. (2007). Mol. Microbiol. 64 : 232-244. Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage λ.

Atsumi, S. and Little, J.W. (2006). A synthetic phage λ regulatory circuit. Proc. Natl. Acad. Sci. USA: 103 : 19045-19050.

Atsumi, S. and Little, J.W. (2006). Analysis of the phage λ gene regulatory circuit by module replacement: Role of the lytic repressor in prophage induction. Proc. Natl. Acad. Sci. USA: 103: 4558-4563.

Michalowski, C.B. and Little, J.W. (2005). Positive autoregulation of cI is a dispensable feature of the phage λ gene regulatory circuitry. J. Bacteriol. 187: 6430-6442.

Michalowski, C.B., Short, M.D. and Little, J.W. 2004. Sequence tolerance of the phage λ pRM promoter: Implications for evolution of gene regulatory circuitry. J. Bacteriol. 186: 7988-7999.

Atsumi, S. and Little, J.W. 2004. Regulatory circuit design and evolution using phage λ. Genes Dev. 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.

Little, J.W., D.P. Shepley, and D.W. Wert. 1999. Robustness of a gene regulatory circuit. EMBO J. 18: 4299-4307.