Publications
Publications
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 (Ptashne, 1992). 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? Several
studies (Little et al., 1999) suggests that the switch can be
simplified by removing various features and still exhibit
qualitatively normal behavior. This evidence 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, but does not carry out the
actual chemistry of cleavage; instead, it acts indirectly as a
"co-protease" to stimulate autodigestion. Hence, the structure of
LexA is designed to allow specific self-cleavage, but to restrain
this reaction in the absence of a stimulus. Studies on mutant
proteins with increased rates of cleavage led to a model in which
LexA exists in two forms. In collaboration with Prof. Natalie
Strynadka, we have recently determined the
structure of several mutant forms of LexA (Luo et al., 2001).
This work reveals two conformations of LexA (see figure). In the
non-cleavable form, the cleavage site (shown in red in spacefill)
lies 20 Å away from the active site (shown in white in
spacefill); in the cleavable form, the red part of the protein moves,
placing the cleavage site in the active site. We believe that RecA
stabilizes the cleavable conformation, leading to rapid cleavage.
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Ptashne, M. (1992). A Genetic Switch: Phage l and Higher Organisms. Cell Press, Cambridge, MA
Lambda regulatory circuitry:
Little J.W., Shepley D.P. and Wert D.W. 1999. Robustness of a gene regulatory circuit. EMBO J. 18: 4299-4307.
Atsumi, S. and Little, J.W. (2004). Regulatory circuit design and evolution using phage l. Genes Dev. 18: 2086-2094.
Michalowski, C.B., Short, M.D. and Little, J.W. (2004). Sequence tolerance of the phage l PRM promoter: Implications for evolution of gene regulatory circuitry. J. Bacteriol. 186: 7988-7999.
Little, J.W. (2005). Threshold effects in gene regulation: When some is not enough. Proc. Natl. Acad. Sci. USA 102: 5311-5312.
Michalowski, C.B. and Little, J.W. (2005). Positive autoregulation of cI is a dispensable feature of the phage l gene regulatory circuitry. J. Bacteriol. 187: 6430-6442.
Atsumi, S. and Little, J.W. (2006). Role of the lytic repressor in prophage induction of phage l as analyzed by a module-replacement approach. Proc. Natl. Acad. Sci. USA: 103: 4558-4563.
Atsumi, S. and Little, J.W. (2006). A synthetic phage l regulatory circuit. Proc. Natl. Acad. Sci. USA: 103: 19045-19050.
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 l."
Crystal structure of LexA:
Luo, Y., et al. (2001). Crystal structure of LexA: A conformational switch for regulation of self-cleavage. Cell 106: 585–594.