Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Revzin, Arnold; Hippel, Peter H. von

doi:10.1021/bi00641a002

Cited by 169 publications

(97 citation statements)

References 33 publications

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“…Our in vivo results are also consistent with in vitro measurements which indicate that the association constant for inducer-saturated repressor to nonspecific DNA sites (KRID) is the same as KRD (9,11,12). Fig.…”

Section: Discussionsupporting

confidence: 90%

“…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)(10)(11)(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).…”

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confidence: 99%

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

Kao-Huang

Revzin

Butler

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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153

105

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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)

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Section: Discussionsupporting

confidence: 90%

mentioning

confidence: 99%

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

Kao-Huang

Revzin

Butler

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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153

105

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“…The absorbance of the rho-RNA complex is separated into protein and nucleic acid contributions by including a control cell in the rotor that contains only rho at the same concentration as in the experimental cell. In this way we have been able to quantitate the concentration of bound and free RNA as a function of position within the ultracentrifuge cell (see Revzin & von Hippel, 1977).…”

Section: Resultsmentioning

confidence: 99%

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

1992

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The rho protein of Escherichia coli interacts with the nascent RNA transcript while RNA polymerase is paused at specific rho-dependent termination sites on the DNA template, and (in a series of steps that are still largely undefined) brings about transcript termination at these sites. In this paper we characterize the interactions of rho with RNA and relate these interactions to the quaternary structure of the functional form of rho. We use CD spectroscopy and analytical ultracentrifugation to determine the binding interactions of rho with RNA ligands of defined length ([rC], where n 2 6). Rho binds to long RNA chains as a hexamer characterized by D3 symmetry. Each hexamer binds -70 residues of RNA. We show by ultracentrifugation and dynamic laser light scattering that, in the presence of RNA ligands less than 22 nucleotide residues in length, rho changes its quaternary structure and becomes a homogeneous dodecamer. The dodecamer contains six strong binding sites for short RNA ligands: i.e., one site for every two rho protomers. The measured association constant of these short RNAs to rho increases with increasing (rC), length, up to n = 9, suggesting that the binding site of each rho protomer interacts with 9 RNA nucleotide residues. Oligo(rC) ligands bound to the strong RNA binding sites on the rho dodecamer do not significantly stimulate the RNA-dependent ATPase activity of rho. Based on these features of the rho-RNA interaction and other experimental data we propose a molecular model of the interaction of rho with its cofactors.

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“…Long-range electrostatic forces bring protein and DNA into proximity, and charged groups of the protein displace cations and water molecules from DNA. This process is accompanied by a large positive entropy change [26,[28][29][30]. Even in the case of the association of the homeodomain with the specific DNA, similar phenomena should happen, thus resulting in an increase in entropy.…”

Section: Dlnka/d(1/t) = -Ah/rmentioning

confidence: 99%

Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

Seimiya¹,

Kurosawa²

1996

FEBS Letters

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The kinetics of binding of the Antp homeodomain to three kinds of DNA fragment were analyzed by measurements of surface plasmon resonance at various temperatures. Non-specific and specific binding of the homeodomain to DNA was examined. In the case of non-specific binding, the association rate constant (kass) was estimated to be 1.41-2.62X 105 M -1 s -1 and the dissociation rate constant (kdiss) was 1.36-3.10 X 10 -2 s -1, thus, the dissociation constant (KI)) was 0.847-1.72 × 10 7 M. The association seemed to be driven by entropy. In the case of specific binding, by contrast, the enthalpy term seemed to contribute more to the binding than did the entropy term. The kass was 2.04-2.59X105 M -1 s -1 and the kdiss was 0.759-1.16X10 -3 s -1, thus, the /to was 2.93-5.69X 10 -9 M. These values were measured under the condition of 150 mM NaCl. Both interactions were strongly dependent on the concentration of NaC1. The /to at 50 mM NaCI became several tens of times smaller than those at 150 mM. Possible reasons for the differences between non-specific and specific interactions are discussed.

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Direct measurement of association constants for the binding of Escherichia coli lac repressor to non-operator DNA

Cited by 169 publications

References 33 publications

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

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

Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA

Kinetics of binding of Antp homeodomain to DNA analyzed by measurements of surface plasmon resonance

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