Kinetic studies on Cro repressor-operator DNA interaction.

The six operators of phage lambda and their consensus sequence were synthesized as 21 base-pair DNAs and their interactions with Cro repressor were studied using a filter binding assay. The measured equilibrium dissociation constants suggest that Cro has the highest affinity to the consensus operator (KD = 1.2 X 10(-12) M) and then the OR3 operator (KD = 2.0 X 10(-12) M), after that the affinity becomes lower in the following order: OR1, OL1, OL2, OL3, OR2. The competition experiments show that Cro forms the most stable complex with the consensus operator (t1/2 = 150 min), which is followed by the complex with OR3 (t1/2 = 70 min), OR1, OL1, OL2, OL3 and OR2. The association rate constants (ka) were also measured. They are approximately the same (2 X 10(8) to 4 X 10(8) m-1 s-1) for the consensus, OR3, OR2 and OR1 operators. These experiments have thus shown that the sequence difference in the operator affects the dissociation (KD and kd) but not the association (ka) process. The operators' binding strengths relative to OR1 are 14 (for consensus operator), 7.6 (OR3), 0.73 (OL1), 0.42 (OL2), 0.16 (OL3) and 0.1 (OR2). Seven different lengths of OR-containing DNA fragments were prepared. Measurement of kinetic parameters shows that the affinity of Cro to operator DNA (measured by KD) is essentially constant and independent of the DNA length, while the association and dissociation rate constants increase as the DNA length increases. This is consistent with the idea that Cro locates and leaves its operator via a two-step mechanism. It appears that Cro binds first at an arbitrary site on DNA, then is transferred to its operator site by a facilitated mechanism. The process is reversed when Cro dissociates from the operator. Most of our data fit to the theoretical expression formulated by Berg, Winter & von Hippel for the sliding mechanism. We conclude that Cro slides along the DNA to locate and leave the operator.