Separation and Characterization of Protein–DNA Complexes by EMSA and In-Gel Footprinting

Charlier, Daniël; Bervoets, Indra

doi:10.1007/978-1-0716-2413-5_11

Cited by 3 publications

(4 citation statements)

References 30 publications

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“…Labeled DNA fragment was incubated with or without HigA2 in 20 mM Tris pH 8 and 200 mM sodium chloride for 1 h, separated using EMSA, and treated in gel with the copper-phenanthroline ion. The reaction products are separated by gel electrophoresis in denaturing conditions along with the corresponding sequencing ladders 45 .…”

Section: Methodsmentioning

confidence: 99%

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae

Hadži,

Živič,

Kovačič

et al. 2024

Nat Commun

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Disordered protein sequences can exhibit different binding modes, ranging from well-ordered folding-upon-binding to highly dynamic fuzzy binding. The primary function of the intrinsically disordered region of the antitoxin HigA2 from Vibrio cholerae is to neutralize HigB2 toxin through ultra-high-affinity folding-upon-binding interaction. Here, we show that the same intrinsically disordered region can also mediate fuzzy interactions with its operator DNA and, through interplay with the folded helix-turn-helix domain, regulates transcription from the higBA2 operon. NMR, SAXS, ITC and in vivo experiments converge towards a consistent picture where a specific set of residues in the intrinsically disordered region mediate electrostatic and hydrophobic interactions while “hovering” over the DNA operator. Sensitivity of the intrinsically disordered region to scrambling the sequence, position-specific contacts and absence of redundant, multivalent interactions, point towards a more specific type of fuzzy binding. Our work demonstrates how a bacterial regulator achieves dual functionality by utilizing two distinct interaction modes within the same disordered sequence.

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Section: Methodsmentioning

confidence: 99%

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae

Hadži,

Živič,

Kovačič

et al. 2024

Nat Commun

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“…Electrophoretic mobility shift assays (EMSAs) were performed as described (Charlier & Bervoets, 2022 ). Either the forward or the reverse primer was radioactively labeled, using fresh γ‐ 32 P‐ATP (Perkin Elmer) and T4 polynucleotide kinase (Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

“…“In‐gel” Cu‐OP footprinting was performed as described (Charlier & Bervoets, 2022 ). First, an EMSA experiment was performed with each reaction containing 300 cps of 32 P‐labeled promoter fragment (bottom strand labeled) (Table A4 ) and an Ah‐BarR octameric protein concentration of 0, 108, and 216 nM.…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms of regulation by a β‐alanine‐responsive Lrp‐type transcription factor from Acidianus hospitalis

Bernauw,

Crabbe,

Ryssegem

et al. 2023

MicrobiologyOpen

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The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid β-alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah-BarR.Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah-BarR is a dual-function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah-BarR protein. β-alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C-terminal tail in Ah-BarR. By performing site-directed mutagenesis, this tail is shown to be involved in ligand-binding response.

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“…El EMSA se desarrolló originalmente para estudiar la unión de proteínas al ADN con secuencias de ADN diana (Fried & Crothers 1981). Esta técnica generalmente usa geles de poliacrilamida (Seo et al, 2019), pero también han sido desarrollados protocolos más rápidos con geles de agarosa (Ream et al, 2016) o combinación con otras técnicas (Charlier & Bervoets, 2022).…”

Section: Electroforesis De Proteínasunclassified

Relación Estructura–función De Las Proteínas. Uso De La Técnica De Electroforesis en El Estudio De Su Expresión

Fuchs Delgado

2022

ASI

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Las proteínas son componentes del metabolismo celular presentes en todos los organismos vivos, donde realizan importantes funciones que están relacionadas con su estructura. Estos compuestos han sido estudiados mediante diferentes técnicas y métodos, pero en los últimos años con el uso de nuevas técnicas, se ha ido cambiando el paradigma sobre estructura-función. En este estudio se realizó un análisis de los nuevos conocimientos sobre la estructura de las proteínas: los plegamientos, motivos y dominios que explican parte de la funcionalidad, las proteínas intrínsicamente desordenadas, también llamadas PINEs o PIDs y las proteínas multifuncionales o Moonlighting, un concepto más flexible en el cual la estructura se adapta a las funciones que realiza. Se revisó el aporte de las nuevas técnicas bioinformáticas en el análisis y organización de una gran cantidad de información que se va produciendo con el estudio de la estructura, mediante cristalografía de rayos X y resonancia magnética nuclear (RMN). También, se analizó el estudio de la expresión de las proteínas a través de la técnica de electroforesis, que continúa siendo una herramienta valiosa y complementaria, para comprender el efecto de los factores ambientales sobre el desarrollo de los organismos.

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Separation and Characterization of Protein–DNA Complexes by EMSA and In-Gel Footprinting

Cited by 3 publications

References 30 publications

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae

Fuzzy recognition by the prokaryotic transcription factor HigA2 from Vibrio cholerae

Molecular mechanisms of regulation by a β‐alanine‐responsive Lrp‐type transcription factor from Acidianus hospitalis

Relación Estructura–función De Las Proteínas. Uso De La Técnica De Electroforesis en El Estudio De Su Expresión

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