How does trp repressor bind to its operator?

The thermodynamics of L-tryptophan and operator DNA binding to the tryptophan repressor of Escherichia coli were analyzed by titration microcalorimetry and van't Hoff analysis of footprinting titrations, respectively. At 25 degrees C in 10 mM sodium phosphate, pH 7.6, and 0.1 M NaCl, the binding of L-tryptophan to the repressor is characterized by values of delta G degrees = -6.04, delta H degree = -14.7, and T delta S degree = -8.67 kcal/mol. The temperature dependence of delta H degree yields delta Cp degree = -0.46 +/- 0.08 kcal/(mol.K) per dimer. The binding is noncooperative at all temperatures studied. At 23 degrees C in 2.5 mM sodium phosphate, pH 7.6, and 25 mM NaCl, the binding of operator DNA to the repressor is characterized by values of delta G degree = -13.3 kcal/mol, delta H degree = -1.55 kcal/mol, T delta S degree = 11.8 kcal/mol, and delta Cp degree = -0.54 +/- 0.10 kcal/(mol.K). Changes in water-accessible surface areas upon binding of L-tryptophan or DNA were calculated from X-ray crystal structures. The experimentally observed delta Cp degree values were compared with delta Cp degree values calculated according to several methods based on various proposed relationships between surface area changes and heat capacity changes. Regardless of which method is used, we find poor agreement between the calorimetric results for L-tryptophan binding and the surface areas calculated from X-ray data; the direction of the discrepancy is that the X-ray data underestimate the value of delta Cp degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Thermodynamics of ligand binding to trp repressor

Jin¹,

Yang²,

Carey³

1993

“…The presence of the corepressor L-tryptophan enhances the affinity of the repressor for the operator by initiating a conformational change to permit the interaction of the sequence-reading helices with two adjacent major grooves of the 18 base pair operator (Zhang et al, 1987). Solution assays including a phosphatase protection assay (Marmorstein et al, 1991) and gel retardation assays (Carey et al, 1991;Haran et al, 1992) both indicate high-affinity binding between the repressor and the operator sequence. However, the nature of the interactions at the interface between the protein and the nucleic acid as implicated by the crystal structures is unusual in that only a single direct amino acid-nucleobase contact appears to be present in each half-site: a bidentate interaction between Arg 69 and the base residue G -9 .…”

mentioning

confidence: 99%

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Smith

McLaughlin

1997

Fourteen analogue DNA sequences containing the trp operator sequence and a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer building block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer is present. These analogue sequences can be used effectively to probe for interactions to either of the prochiral phosphate oxygens as illustrated by their use to identify critical interactions in the trp repressor-operator complex. In a number of cases, the pairs of diastereomeric analogue sequences exhibit variable binding affinities that can be used to identify one of the prochiral phosphate oxygens as a critical site for complex-stabilizing interactions. Upon the basis of dissociation constants, apparent incremental binding energies are assigned to specific interactions. In all but one example, these identified sites for interactions to the phosphate backbone can be correlated with contacts implicated by the crystal structure analysis of the trp repressor-operator complex.

“…When TR binds this site, it represses transcription of those genes whose protein products are responsible for the synthesis of tryptophan. The repressor protein has been extensively studied from a genetic as well as a structural standpoint (Kelley & Yanofsky, 1985 in inducing specific DNA binding is still not well understood (Carey et al, 1991;Fernando & Royer, 1992).…”

mentioning

confidence: 99%

Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding

LeTilly

Royer²

1993

161

112

The study of interactions between proteins and nucleic acids is central to the understanding of the control of genetic expression. Fluorescence anisotropy has been used to measure, in solution, the equilibrium binding profiles of a bacterial repressor protein, the tryptophan repressor (TR), to a fluorescently labeled oligonucleotide containing one of its target operator sequences. Investigation of the effects of changing concentrations of corepressor, operator DNA, and protein implicate TR oligomers in the regulation of DNA binding. These studies also demonstrate that the relatively straightforward technique of fluorescence anisotropy can be applied to the study of the interactions between proteins and nucleic acids. The fluorescence technique exhibits sufficient sensitivity to replace radioactive methods of detection in most cases. In addition, since it is a solution-based methodology, it offers a true equilibrium measure of the protein-nucleic acid equilibria, and the effects of changes in solution conditions such as salt and ligand concentration, pH, and temperature can be readily evaluated. Data acquisition is relatively simple and rapid, and the data are of sufficient quality for detailed thermodynamic analyses of complex systems. Given these attributes, fluorescence anisotropy will find multiple applications in the area of genetic regulation.