ABSTBACr Binding of genome regulatory proteins to nonspecific DNA sites may play an important role in controlling the thermodynamics and kinetics of the interactions of these proteins with their specific target DNA sequences. An estimate of the fraction of Escherchia colilac repressor molecules bound in vivo to the operator region and to nonoperator sites on the E coli chromosome is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678-54) and the DNA-free minicells derived therefrom. Assuming the minicell cytoplasm to be representative of that of the parent E. coli cells, we find that less than 10% of the repressor tetramers of the average cell are free in solution; the remainder are presumed to be bound to the bacterial chromosome. The minimum in vivo value of the association constant for repressor to bulk nonoperator DNA (Kim) (1-4).The lactose operon of Escherichia coli is currently the best molecularly characterized genome regulatory system. The lac repressor controls the level of expression of the lac genes by binding tightly to the operator region and preventing transcription of the operon by DNA-dependent RNA polymerase (5-7). The affinity of repressor for operator in vitro, and the level of repression of the operon in vivo, are allosterically modulated by the binding to repressor of small molecule inducers structurally related to allolactose, the "natural" inducer which occurs as an early intermediate in the metabolic degradation of lactose (5, 8). Nonoperator DNA also binds lac repressor, albeit much more weakly than does operator (9-12), and this nonspecific affinity is not changed by the binding of inducer to the repressor (9, 11, 12). Furthermore, RNA polymerase also binds to DNA sites other than promoters, and its affinity for nonpromoter sequences may be modified by interaction with a factor (1, 4, 13). Based on these observations and direct in vitro measurement of most of the relevant binding constants, a model has been developed to account quantitatively for the observed levels of repression and derepression of the lactose operon; this model includes, as a central feature, nonspecific binding of regulatory proteins to the E. coli chromosome (1, 11).The validity of such models is not crucially dependent on knowledge of the absolute binding constants of (e.g.) the lac repressor (R) and repressor-inducer complex (RI) to operator (0) and nonoperator DNA (D)