The Solution Structures of the trp Repressor-Operator DNA Complex

Zhang, Hong; Zhao, Daqing; Revington, Matthew; Lee, Weontae; Jia, Xu; Arrowsmith, C.H.; Jardetzky, Oleg

doi:10.1006/jmbi.1994.1317

Cited by 117 publications

(98 citation statements)

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“…Because of the degeneracy of the Hy resonances (1.62 ppm), separation of the NOES via the 13Cy shifts is necessary to make the unambiguous assignments indicated. The close proximity of these two residues to both the DNA and the corepressor render these intermolecular NOES crucial for a detailed structural description of the complex [6]. On the basis of the experiment described above, a total of 28 intermolecular NOES were unambiguously identified.…”

Section: Resultsmentioning

confidence: 99%

“…The sample used for NMR consisted of 1.0 mM uniformly "C enriched trp-repressor dimer, 2.0 mM unlabeled tryptophan, I.0 mM unlabeled operator DNA [6], 99.9% D,O, pH 6.0, 50 mM potassium phosphate buffer, 37°C. The 3D 13C F,-edited,F,-filtered HMQC-NOESY spectrum was recorded as an 84 x 26 x 5 12 complex matrix on a Varian UNITYplus 500 MHz spectrometer equipped with a gradient unit and an actively shielded triple resonance probehead.…”

Section: Methodsmentioning

confidence: 99%

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A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

et al. 1994

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A pulsed field gradient three-dimensional isotope-filtered "C HMQC-NOESY experiment has been developed to characterize intermolecular contacts in a 37 kDa macromolecular ternary complex consisting of uniformly "C labeled trp-repressor, its natural abundance co-repressor, L-tryptophan, and natural abundance operator DNA. The pulse scheme makes use of pulsed field gradients for the removal of artifacts and dephasing of unwanted magnetization during isotope filtering, and employs a strategy to minimize the time that magnetization resides in the transverse plane. The experiment provides solely intermolecular NOE contacts between protons of the labeled protein and protons of the unlabeled species, and has proven to be especially useful in eliminating ambiguities between intra-and intermolecular NOES in the isotope-edited 3D '% HMQC-NOESY spectrum of the complex.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

et al. 1994

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“…Its structure in both the Trp-free apo-form and the Trp-bound holo-form, and the structure of its DNA complexes, have been determined by both X-ray and NMR analyses (Schevitz et al, 1985;Otwinowski et al, 1988;Lawson & Carey, 1993;Zhao et al, 1993;Zhang et al, 1994). The repressor is known to interact with at least five different operator sequences, i.e., in the trp, trpR, aroH, aroL, and mtr operons Reprint requests to: Oleg Jardetzky, Stanford Magnetic Resonance Laboratory, Stanford University, Stanford, California 94305.5055; e-mail: jardetzky@camis.stanford.edu.…”

Section: " " ~~~mentioning

confidence: 99%

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

et al. 1996

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Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.

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“…of sequence-specific contacts with the DNA bases (Sauer et al, 1990;Harrison and Aggarwal, 1990), such specific interactions are virtually absent in the crystal structure of the trp repressor-operator complex (Otwinowslu et al, 1988 ;Luisi and Sigler, 1990); the situation in solution is not yet clear (Zhang et al, 1994). In the crystal, most of the contacts are made to the phosphate backbone of the DNA, although there are some structurally significant water-mediated contacts between the protein backbone and mutationally sensitive bases.…”

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The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide

Ramesh

Frederick

Syed

et al. 1994

European Journal of Biochemistry

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The effects of the binding of the corepressor l‐tryptophan and an operator oligonucleotide to Escherichia coli trp repressor have been studied, using selective 15N labelling to permit observation of the backbone amide resonances of 50 of the 107 residues of the protein monomer. Repressor molecules selectively labelled in turn with [15N]alanine, [15N]glutamate, [15N]isoleucine, [15N]leucine and [15N]methionine were prepared by isolating them from prototrophic E. coli cells grown in media containing a mixture of unlabelled and the appropriate 15N‐enriched amino acids. Analysis of the heteronuclear correlation spectra of the labelled repressors shows the value of selective labelling in resolving the crosspeaks of, for example, the 19 leucine and 12 glutamate residues. All 50 residues studied show measurable changes in amide 1H and/or 15N chemical shift on the binding of tryptophan and/or the operator oligonucleotide, showing clearly that ligand binding has effects which are transmitted throughout almost the whole protein. Large chemical shift changes on ligand binding are seen in residues in the tryptophan binding site and in the ‘helix‐turn‐helix’ DNA‐binding domain, but also in residues in helices C and F remote from the ligand binding sites. On operator binding there is selective broadening of the signals of residues in the N‐terminal region of the protein and in the DNA‐binding domain, perhaps reflecting a conformational equilibrium.

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The Solution Structures of the trp Repressor-Operator DNA Complex

Cited by 117 publications

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A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide

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The Solution Structures of the trp Repressor-Operator DNA Complex

Cited by 117 publications

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A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

A pulsed field gradient isotope‐filtered 3D 13C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

Flexibility of dna binding domain of trp repressor required for recognition of different operator sequences

The interactions of Escherichia coli trp repressor with tryptophan and with an operator oligonucleotide

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A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes

A pulsed field gradient isotope‐filtered 3D ¹³C HMQC‐NOESY experiment for extracting intermolecular NOE contacts in molecular complexes