Robustness of a gene regulatory circuit.

Complex interacting systems exhibit system behavior that is often not predictable from the properties of the component parts. We have tested a particular system property, that of robustness. The behavior of a system is termed robust if that behavior is qualitatively normal in the face of substantial changes to the system components. Here we test whether the behavior of the phage lambda gene regulatory circuitry is robust. This circuitry can exist in two alternative patterns of gene expression, and can switch from one regulatory state to the other. These states are stabilized by the action at the O(R) region of two regulatory proteins, CI and Cro, which bind with differential affinities to the O(R)1 and O(R)3 sites, such that each represses the synthesis of the other one. In this work, this pattern of binding was altered by making three mutant phages in which O(R)1 and O(R)3 were identical. These variants had the same qualitative in vivo patterns of gene expression as wild type. We conclude that the behavior of the lambda circuitry is highly robust. Based on these and other results, we propose a two-step pathway, in which robustness plays a key role, for evolution of complex regulatory circuitry.

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.