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

Abstract

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)

DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae.

“…This result led them also to conclude that DNA binding and localization in the nucleus are independent of one another. In E. coli, almost all of the lac repressor is nonspecifically associated with DNA (11). If LexA has a nonspecific affinity for DNA comparable to that of the lac repressor, it is not unreasonable to expect all of LexA in S. cerevisiae to be associated with nuclear DNA, if LexA has free access to every compartment in the cell.…”

Transcriptional regulation by the numbers: models

“…For the purposes of this review, we make the simplified assumption that the key molecular players (RNAP and TFs) are bound to the DNA either specifically or non-specifically. This question has been addressed in the context of the λ switch [27], for the lac repressor [21,28] and for RNAP [29]. Stated differently, as a simplification, we will ignore the contribution of 'free' polymerase in the cytoplasm, in addition to those RNAP molecules that are engaged in transcription on other promoters.…”

Models of transcription factor binding: Sensitivity of activation functions to model assumptions

“…They find the specific sites through repeated binding and unbinding to/from non-specific sites, rather than through direct attachment from an unbound state. In cells a certain proportion of TFs will normally be freely suspended in the cytoplasm (Kao-Huang et al, 1977;Wunderlich and Mirny, 2008). It feels safe to ignore this effect given that it seems unlikely that a TF directly binds to the specific site from a suspended state in the cytoplasm.…”