Mean DNA Bend Angle and Distribution of DNA Bend Angles in the CAP-DNA Complex in Solution

doi:10.1006/jmbi.2001.4976

Journal of Molecular Biology

Volume 312, Issue 3, 21 September 2001, Pages 453-468

https://doi.org/10.1006/jmbi.2001.4976 Get rights and content

Abstract

In order to define the mean DNA bend angle and distribution of DNA bend angles in the catabolite activator protein (CAP)-DNA complex in solution under standard transcription initiation conditions, we have performed nanosecond time-resolved fluorescence measurements quantifying energy transfer between a probe incorporated at a specific site in CAP, and a complementary probe incorporated at each of five specific sites in DNA. The results indicate that the mean DNA bend angle is 77(±3)° – consistent with the mean DNA bend angle observed in crystallographic structures (80(±12)°). Lifetime-distribution analysis indicates that the distribution of DNA bend angles is relatively narrow, with <10 % of DNA bend angles exceeding 100 °. Millisecond time-resolved luminescence measurements using lanthanide-chelate probes provide independent evidence that the upper limit of the distribution of DNA bend angles is ∼100 °. The methods used here will permit mutational analysis of CAP-induced DNA bending and the role of CAP-induced DNA bending in transcriptional activation.

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^☆: Abbreviations used: CAP, catabolite activator protein; CRP, cAMP receptor protein; 1, 5-IAEDANS, 5-[(iodoacetamido-ethyl)-amino]naphthalene-1-sulfonic acid; AEDANS, 5-[(acetamido-ethyl)-amino]napthalene-1-sulfonic acid; FRET, fluorescence resonance energy transfer; LRET, luminescence resonance energy transfer; FWHM, full width at half maximum

^☆☆: Edited by P. E. Wright

^f1: Present address: A. N. Kapanidis, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

^f2: Corresponding author

^f3: E-mail address of the corresponding author: [email protected]

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Journal of Molecular Biology

Regular ArticleMean DNA Bend Angle and Distribution of DNA Bend Angles in the CAP-DNA Complex in Solution☆,☆☆

Abstract

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Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Curr. Opin. Struct. Biol.

Mol. Cell

Biophys. J.

Biophys. J.

J. Comput. Phys.

Transcriptional regulation by cAMP and its receptor protein

Ann. Rev. Biochem.

Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site

Nucl. Acids Res.

Crystal structure of a CAP-DNA complex: the DNA is bent by 90o

Science

Aromatic hydrogen bond in sequence-specific protein DNA recognition

Nature Str. Biol.

The structure of a CAP-DNA complex having two cAMP molecules bound to each other

Proc. Natl Acad. Sci. USA

Transcriptional activation by Escherichia coli CAP protein

DNA sequence determinants of CAP-induced bending and protein binding affinity

Nature

The locus of sequence-directed and protein-induced DNA bending

Nature

Empirical estimation of protein-induced DNA bending angles: applications to λ site-specific recombination complexes

Nucl. Acids Res.

Comparative gel electrophoresis measurement of the DNA bend angle induced by the catabolite activator protein

Biopolymers

Identification of the activating region of catabolite gene activator protein (CAP): isolation and characterization of mutants of CAP specifically defective in transcription activation

Proc. Natl Acad. Sci. USA

The change of DNA structure by specific binding of the cAMP receptor protein from rotation diffusion and dichroism measurements

EMBO J.

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J. Biomol. Struct. Dynam

Variations of intramolecular ligation rates allow the detection of protein-induced bends in DNA

EMBO J.

Protein-induced bending and DNA cyclization

Proc. Natl Acad. Sci. USA

Solution studies of the structure of bent DNA in the cAMP receptor protein-lac DNA complex

Biochemistry

Regular Article
Mean DNA Bend Angle and Distribution of DNA Bend Angles in the CAP-DNA Complex in Solution☆,☆☆

Crystal structure of a CAP-DNA complex: the DNA is bent by 90^o