Methods to Quantitatively Measure Topological Changes Induced by DNA-Binding Proteins In Vivo and In Vitro.

Agarose gel electrophoresis in the presence of chloroquine (an intercalating agent) can be used to resolve and characterize the population of topoisomers present in supercoiled plasmid DNA. Here, we describe how chloroquine gel electrophoresis can capture changes in the topoisomer distribution of plasmid DNA that bears a recognition site for a given protein, if that plasmid is isolated from cells producing the protein of interest. We also describe two complementary in vitro assays, which can be used to capture transient changes in DNA supercoiling caused when the purified protein of interest engages its recognition site.

VirB, a key transcriptional regulator of virulence, requires a CTP ligand for its regulatory activities.

species cause bacillary dysentery, the second leading cause of diarrheal deaths worldwide. There is a pressing need to identify novel molecular drug targets. virulence phenotypes are controlled by the transcriptional regulator, VirB.

VirB, a key transcriptional regulator of virulence, requires a CTP ligand for its regulatory activities.

Unlabelled: The VirB protein, encoded by the large virulence plasmid of spp., is a key transcriptional regulator of virulence genes. Without a functional gene, cells are avirulent.

Localized modulation of DNA supercoiling, triggered by the Shigella anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In Shigella spp., at ≤30°C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid.

Localized modulation of DNA supercoiling, triggered by the anti-silencer VirB, is sufficient to relieve H-NS-mediated silencing.

In Bacteria, nucleoid structuring proteins govern nucleoid dynamics and regulate transcription. In ., at ≤ 30 °C, the histone-like nucleoid structuring protein (H-NS) transcriptionally silences many genes on the large virulence plasmid.