Nonspecific DNA binding of genome-regulating proteins as a biological control mechanism: Measurement of DNA-bound Escherichia coli lac repressor in vivo

Abstract

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 Escherichia coli lac 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 ( K RD ) calculated from these results is about 10 3 M -1 , and analysis of the sources of error in the minicell experiment suggests that the actual in vivo value of K RD could be substantially greater. The value of K RD , coupled with in vitro data on the ionic strength dependence of this parameter, can be used to estimate that the effective intracellular cation activity of E. coli is no greater than about 0.24 M (and probably no less than 0.17 M) in terms of sodium ion equivalents. The minicell distribution experiments also confirm that the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA ( K RID ) is [unk] K RD in vivo . These results are used to calculate minimum in vivo values of K RO and K RIO (association constants for repressor and for inducer-repressor complex binding to operator) of about 10 12 M -1 and about 10 9 M -1 , respectively. The results fit a quantitative model for operon regulation in which nonspecific DNA-repressor complexes play a key role in determining basal and constitutive levels of gene expression [von Hippel, P. H., Revzin, A., Gross, C. A. & Wang, A. C. (1974) Proc. Natl. Acad. Sci. USA 71, 4808-4812].