Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro

Ebright, Yon W.; Chen, Yan; Pendergrast, P. S.; Ebright, Richard H.

doi:10.1021/bi00159a004

Cited by 107 publications

(96 citation statements)

References 51 publications

(57 reference statements)

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“…In the case of EDTA-conjugation to proteins, disulfide bond linkage to a Cys side-chain is commonly used (9,(41)(42)(43). As the reaction is reversible, the conversion rate cannot be 100% and there is always a small percentage of unconverted diamagnetic species.…”

Section: Considerations Of Sample Stability For 1 H-γ 2 Measurementsmentioning

confidence: 99%

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Iwahara

Tang

Clore

2007

Journal of Magnetic Resonance

242

385

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The use of 1 H transverse paramagnetic relaxation enhancement (PRE) has seen a resurgence in recent years as method for providing long-range distance information for structural studies and as a probe of large amplitude motions and lowly populated transient intermediates in macromolecular association. In this paper we discuss various practical aspects pertaining to accurate measurement of PRE 1 H transverse relaxation rates (Γ 2 ). We first show that accurate Γ 2 rates can be obtained from a two time-point measurement without requiring any fitting procedures or complicated error estimations, and no additional accuracy is achieved from multiple time-point measurements recorded in the same experiment time. Optimal setting of the two time-points that minimize experimental errors is also discussed. Next we show that the simplistic single time-point measurement that has been commonly used in the literature, can substantially underestimate the true value of Γ 2 , unless a relatively long repetition delay is employed. We then examine the field dependence of Γ 2 , and show that Γ 2 exhibits only a very weak field dependence at high magnetic fields typically employed in macromolecular studies. The theoretical basis for this observation is discussed. Finally, we investigate the impact of contamination of the paramagnetic sample by trace amounts (≤5%) of the corresponding diamagnetic species on the accuracy of Γ 2 measurements. Errors in Γ 2 introduced by such diamagnetic contamination are potentially sizeable, but can be significantly reduced by using a relatively short time interval for the two time-point Γ 2 measurement.

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Section: Considerations Of Sample Stability For 1 H-γ 2 Measurementsmentioning

confidence: 99%

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Iwahara

Tang

Clore

2007

Journal of Magnetic Resonance

242

385

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“…This approach presents several advantages. The iron-EDTA complex can be stably introduced into the native protein (Ermficora et al 1992} and does not promote spontaneous cleavage of the DNA (Ebright et al 1992;Mazzarelli et al 1993). This permits assembly of higher order nucleoprotein structures that require supercoiled substrates.…”

Section: Conversion Of Hu Into a Chemical Nucleasementioning

confidence: 99%

“…This permits assembly of higher order nucleoprotein structures that require supercoiled substrates. Upon the addition of a reducing agent, DNA cleavage ensues rapidly, is highly localized, and shows little or no sequence specificity (Ebright et al 1992;Mazzarelli et al 1993).…”

Section: Conversion Of Hu Into a Chemical Nucleasementioning

confidence: 99%

Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease.

Lavoie¹,

Chaconas²

1993

Genes & Development

104

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Higher order nucleoprotein complexes mediate a variety of DNA metabolic events such as replication, transcription, recombination, and transposition in both eukaryotic and prokaryotic systems (Echols 1986. The assembly of these structures requires the coordination of multiple protein-protein and protein-DNA interactions and may require the deformation of the DNA helix by melting, wrapping, bending, and/or looping {for review,

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“…Experiments described herein suggest that the CTDs of the two ␣ subunits can interact with different subunits of both bound CRP dimers, and this results in complexes of different architectures depending on the positions of the upstream CRP dimer. To investigate the effects of CRP in positioning of the two ␣ subunits, we have applied an affinity DNA cleavage method by protein-conjugated EDTA⅐Fe (17)(18)(19)(20), which provides information on the location of each of the two ␣ subunits. (20).…”

mentioning

confidence: 99%

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

Murakami

Owens

Belyaeva

et al. 1997

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

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Interactions between the cAMP receptor protein (CRP) and the carboxy-terminal regulatory domain (CTD) of Escherichia coli RNA polymerase ␣ subunit were analyzed at promoters carrying tandem DNA sites for CRP binding using a chemical nuclease covalently attached to ␣. Each CRP dimer was found to direct the positioning of one of the two ␣ subunit CTDs. Thus, the function of RNA polymerase may be subject to regulation through protein-protein interactions between the two ␣ subunits and two different species of transcription factors.The RNA polymerase holoenzyme of Escherichia coli is composed of core enzyme with subunit structure ␣ 2 ␤␤Ј, responsible for RNA polymerization, and one of multiple species of subunit, responsible for promoter recognition. Promoter selectivity of the holoenzyme is modulated by direct or indirect interaction with many transcription factors, resulting in switching of the global pattern of gene transcription according to the environment. The best-characterized target on the RNA polymerase involved in molecular communication with transcription factors is the ␣ subunit carboxy-terminal domain (CTD) that contains the contact sites for class I transcription factors. The ␣ subunit, consisting of 329 amino acid residues, is composed of two structural domains, each responsible for distinct functions (1-3) and each forming independent structural domains connected by a protease-sensitive flexible linker (4-6). The amino (N)-terminal domain from residues 20 to 235 plays a key role in RNA polymerase assembly by providing the contact surface for ␣ dimerization and binding of ␤ and ␤Ј subunits (7-10), whereas the CTD from residues 235 to 329 plays a regulatory role by providing the contact surfaces for trans-acting protein factors and cis-acting DNA elements (11)(12)(13)(14).Whereas the regulation of many E. coli promoters involves a single factor, some promoters are regulated by two or more transcription factors, and such coregulation systems involving multiple species of transcription factors can couple gene expression to diverse environmental conditions. Knowledge of the molecular mechanism of prokaryotic transcription regulation involving more than two factors would contribute much to understanding of the events carried out in eukaryotes, because the regulation of gene transcription in eukaryotes generally involves the action of multiple transcription factors. To gain insight into this problem, we analyzed interactions between RNA polymerase and cAMP receptor protein (CRP) dimers on promoters carrying tandem CRP-binding sites at various positions relative to the transcription start site. A set of promoters was constructed carrying one DNA site for CRP centered at position Ϫ41.5 upstream from the transcription start point and a second DNA site for CRP located further upstream (refs. 15

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Incorporation of an EDTA-metal complex at a rationally selected site within a protein: application to EDTA-iron DNA affinity cleaving with catabolite gene activator protein (CAP) and Cro

Cited by 107 publications

References 51 publications

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Site-specific HU binding in the Mu transpososome: conversion of a sequence-independent DNA-binding protein into a chemical nuclease.

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

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