LINKAGE BETWEEN OPERATOR BINDING AND DIMER TO OCTAMER SELF-ASSEMBLY OF BACTERIOPHAGE-LAMBDA CI REPRESSOR

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators O-R and O-L controls the develop mental state of the phage. Cooperativity has long been thought to be m ediated by self-assembly of repressor dimers to form tetramers which c an bind simultaneously to adjacent operators. More recently, we demons trated that when free repressor dimers self-associate in solution, tet ramer is an intermediate in a concerted assembly reaction leading to o ctamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in th e assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultan eously, octamer could account for three-way cooperativity. In fact, ba sed solely on repressor self-assembly, the naive prediction is that th e repressor-O-R interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to coope rativity from processes other than repressor self-assembly. Here, we f ocus on coupling between repressor self-association and operator bindi ng as one possible unfavorable contribution to cooperativity. Sediment ation equilibrium analysis was used to compare the dimer-octamer assoc iation reactions of a repressor dimer-O(R)1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate O(R)1 binding to free dimers and dimers assembled as higher order species. The resul ts of these experiments indicate a significant and salt-dependent unfa vorable contribution generated by such coupling. Since the oligonucleo tides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechan ism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavo rable contributions generated by structural transitions in both macrom olecules serve to moderate the effect of self-association alone. We sp eculate that this is a general feature of cooperative protein-DNA inte ractions.