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Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.

Jameson KH, Rostami N, Fogg MJ, Turkenburg JP, Grahl A, Murray H, Wilkinson AJ - Mol. Microbiol. (2014)

Bottom Line: Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo.Localization of GFP-SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA.The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK.

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Molecular mass measured from SEC-MALLS analysis. In A and B, the thinner lines trace the differential refractive index of the eluate from a Superdex 10/30 S75 column as a function of time. The thicker lines represent the weight average molecular weight of the species in the eluate, calculated from refractive index and light-scattering measurements.A. Overlay of chromatograms for DnaADI at 3 concentrations: 1 mg ml−1 (green), 2.5 mg ml−1 (red) and 5 mg ml−1 (blue), revealing species of mass 11 kDa indicating that DnaADI is a monomer.B. Overlay of chromatograms for SirA–DnaADI at 3 concentrations: 0.5 mg ml−1 (green), 1.0 mg ml−1 (red) and 2.5 mg ml−1 (blue). The derived Mw values for the principal species are 25–28 kDa indicating that the SirA–DnaADI complex is a 1:1 heterodimer. There is evidently, excess/dissociated DnaADI giving rise to the minor peak eluting at ∼28 min.
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fig01: Molecular mass measured from SEC-MALLS analysis. In A and B, the thinner lines trace the differential refractive index of the eluate from a Superdex 10/30 S75 column as a function of time. The thicker lines represent the weight average molecular weight of the species in the eluate, calculated from refractive index and light-scattering measurements.A. Overlay of chromatograms for DnaADI at 3 concentrations: 1 mg ml−1 (green), 2.5 mg ml−1 (red) and 5 mg ml−1 (blue), revealing species of mass 11 kDa indicating that DnaADI is a monomer.B. Overlay of chromatograms for SirA–DnaADI at 3 concentrations: 0.5 mg ml−1 (green), 1.0 mg ml−1 (red) and 2.5 mg ml−1 (blue). The derived Mw values for the principal species are 25–28 kDa indicating that the SirA–DnaADI complex is a 1:1 heterodimer. There is evidently, excess/dissociated DnaADI giving rise to the minor peak eluting at ∼28 min.

Mentions: The stoichiometry of the SirA–DnaADI complex was determined using size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). In these experiments, samples are fractionated on a gel-filtration column and the absorbance at 280 nm and the refractive index of the eluate are monitored together with the multi-angle laser light scattering of the sample. This enables the weight average molecular weight (Mw) of species in the eluate to be calculated continuously. Samples of DnaADI and SirA–DnaADI were analysed at a series of protein concentrations. As shown in Fig. 1A, DnaADI elutes from the size exclusion column as a single A280 peak at ∼ 27.5 min and has an experimentally determined molecular mass of ∼ 11 kDa. This suggests that DnaADI from B. subtilis is a monomer (calculated molecular mass = 9.7 kDa) in contrast to E. coli DnaADI which is reported to form dimers under similar conditions (Abe et al., 2007).


Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.

Jameson KH, Rostami N, Fogg MJ, Turkenburg JP, Grahl A, Murray H, Wilkinson AJ - Mol. Microbiol. (2014)

Molecular mass measured from SEC-MALLS analysis. In A and B, the thinner lines trace the differential refractive index of the eluate from a Superdex 10/30 S75 column as a function of time. The thicker lines represent the weight average molecular weight of the species in the eluate, calculated from refractive index and light-scattering measurements.A. Overlay of chromatograms for DnaADI at 3 concentrations: 1 mg ml−1 (green), 2.5 mg ml−1 (red) and 5 mg ml−1 (blue), revealing species of mass 11 kDa indicating that DnaADI is a monomer.B. Overlay of chromatograms for SirA–DnaADI at 3 concentrations: 0.5 mg ml−1 (green), 1.0 mg ml−1 (red) and 2.5 mg ml−1 (blue). The derived Mw values for the principal species are 25–28 kDa indicating that the SirA–DnaADI complex is a 1:1 heterodimer. There is evidently, excess/dissociated DnaADI giving rise to the minor peak eluting at ∼28 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285326&req=5

fig01: Molecular mass measured from SEC-MALLS analysis. In A and B, the thinner lines trace the differential refractive index of the eluate from a Superdex 10/30 S75 column as a function of time. The thicker lines represent the weight average molecular weight of the species in the eluate, calculated from refractive index and light-scattering measurements.A. Overlay of chromatograms for DnaADI at 3 concentrations: 1 mg ml−1 (green), 2.5 mg ml−1 (red) and 5 mg ml−1 (blue), revealing species of mass 11 kDa indicating that DnaADI is a monomer.B. Overlay of chromatograms for SirA–DnaADI at 3 concentrations: 0.5 mg ml−1 (green), 1.0 mg ml−1 (red) and 2.5 mg ml−1 (blue). The derived Mw values for the principal species are 25–28 kDa indicating that the SirA–DnaADI complex is a 1:1 heterodimer. There is evidently, excess/dissociated DnaADI giving rise to the minor peak eluting at ∼28 min.
Mentions: The stoichiometry of the SirA–DnaADI complex was determined using size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS). In these experiments, samples are fractionated on a gel-filtration column and the absorbance at 280 nm and the refractive index of the eluate are monitored together with the multi-angle laser light scattering of the sample. This enables the weight average molecular weight (Mw) of species in the eluate to be calculated continuously. Samples of DnaADI and SirA–DnaADI were analysed at a series of protein concentrations. As shown in Fig. 1A, DnaADI elutes from the size exclusion column as a single A280 peak at ∼ 27.5 min and has an experimentally determined molecular mass of ∼ 11 kDa. This suggests that DnaADI from B. subtilis is a monomer (calculated molecular mass = 9.7 kDa) in contrast to E. coli DnaADI which is reported to form dimers under similar conditions (Abe et al., 2007).

Bottom Line: Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo.Localization of GFP-SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA.The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK.

Show MeSH
Related in: MedlinePlus