Comparative observations of biological specimens, especially DNA and filamentous actin molecules in atomic force, tunnelling and electron microscopes

Delain, Etienne; Fourcade, A; Poulin, Jean-Claude; Barbin, Agnès; Coulaud, Dominique; Cam, Eric Le; Paris, Emmanuel

doi:10.1051/mmm:0199200306045700

Cited by 38 publications

(28 citation statements)

References 42 publications

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“…To analyze LrpC/DNA complexes by AFM, 20 l of the same solutions used for EM in presence of 5 mM Mg 2ϩ were deposited onto freshly cleaved mica and then washed with 0.2% (w/v) aqueous uranyl acetate (23). The observation was performed in the tapping mode in air specifically available with nanoscope IIIa (Digital Instruments/Veeco).…”

Section: Methodsmentioning

confidence: 99%

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

Beloin¹,

Jeusset²,

Révet³

et al. 2003

Journal of Biological Chemistry

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The Bacillus subtilis LrpC protein belongs to the Lrp/ AsnC family of transcriptional regulators. It binds the upstream region of the lrpC gene and autoregulates its expression. In this study, we have dissected the mechanisms that govern the interaction of LrpC with DNA by electrophoretic mobility shift assay, electron microscopy, and atomic force microscopy. LrpC is a structurespecific DNA binding protein that forms stable complexes with curved sequences containing phased A tracts and wraps DNA to form spherical, nucleosomelike structures. Formation of such wraps, initiated by cooperative binding of LrpC to DNA, results from optimal protein/protein interactions specified by the DNA conformation. In addition, we have demonstrated that LrpC constrains positive supercoils by wrapping the DNA in a right-handed superhelix, as visualized by electron microscopy.

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Section: Methodsmentioning

confidence: 99%

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

Beloin¹,

Jeusset²,

Révet³

et al. 2003

Journal of Biological Chemistry

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“…Therefore, it is interesting to use both techniques and consider their findings as complementary. This study is one of the first to compare EM/AFM observations of a DNA-protein complex since the combination ofthese two methods is rarely used (10,11). The AFM images of DNA-protein complexes, obtained with the Tapping Mode, considered less aggressive than the tip scan- ning of contact AFM, are among the best available, AFM providing the volumetric information that is lacking with EM.…”

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confidence: 99%

“…Furthermore, negative staining provides additional information about the structural organization of proteins bound to DNA. Due to electron-induced damage ofthe biological specimen with EM, the atomic force microscope (AFM) is now considered an interesting tool for high-resolution imaging of interacting biological macromolecules, especially DNA and proteins (10)(11)(12)(13)(14)(15).…”

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Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

Cam

Frechon

Barray

et al. 1994

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

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“…The probe position was maintained vertical to the sample surface by monitoring the oscillation amplitude damping due to probe-sample interactions. AFM measurements of SpoIIE-loaded DOPC vesicles were taken at room temperature on samples that had condensed to single bilayers on 1-cm 2 plates of mica after overnight incubation at 4°C (15,39). Histograms of the surface height of the lipid bilayers were produced by the University of Texas Health Center, San Antonio, Image Tool software package.…”

Section: Methodsmentioning

confidence: 99%

Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane

et al. 2004

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Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor F , and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.Spore formation by the gram-positive soil bacterium Bacillus subtilis has been extensively studied as a simple example of cellular development and differentiation (21, 67). During vegetative growth of B. subtilis, medial division gives rise to two identical daughter cells, but under conditions of nutritional starvation, the cell forms a large mother cell and a smaller prespore, which develops into a tough, dormant spore. Sporulation in prokaryotes is a complex process, involving an ordered multiprotein division apparatus, which initiates an annular invagination of the cell wall and the formation of an asymmetric septal membrane of two bilayers, separated by peptidoglycan. Differentiation in the prespore is initiated by activation of the transcription factor F , the first sporulationspecific sigma factor in the developmental cascade (21,37,44,63). The sigma factor F and its regulatory proteins SpoIIAA, SpoIIAB, and SpoIIE are synthesized before septation (6, 25, 56), but F remains bound to the anti-sigma factor SpoIIAB, and therefore in an inhibited state, prior to closure of the asymmetric septum (3,19,51). In the predivisional cell, the anti-anti-sigma factor SpoIIAA is phosphorylated at a single site (16, 18, 51, 52). The resulting SpoIIAA-P cannot bind SpoIIAB (the protein kinase for SpoIIAA) to release F from the SpoIIAB-F complex (16,19,51). The phosphatase domain of the 91.5-kDa enzyme SpoIIE is responsible for the hydrolysis of SpoIIAA-P (4, 17, 24). Expression of SpoIIE during the early stages of sporulation does not lead to discernible concentrations of no...

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Comparative observations of biological specimens, especially DNA and filamentous actin molecules in atomic force, tunnelling and electron microscopes

Cited by 38 publications

References 42 publications

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

Contribution of DNA Conformation and Topology in Right-handed DNA Wrapping by the Bacillus subtilis LrpC Protein

Observation of binding and polymerization of Fur repressor onto operator-containing DNA with electron and atomic force microscopes.

Evaluation of the Kinetic Properties of the Sporulation Protein SpoIIE of Bacillus subtilis by Inclusion in a Model Membrane

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