Fig 1.
Schematic diagram showing the experimental procedure followed.
The left panel shows the experiment procedure without reversing the crosslinking between DNA and the protein. In the right panel shows the method followed for control where the cross linking is reversed with Rapigest before harvesting the cells.
Fig 2.
Graph depicting the concentration of protein in eluates when different amount of formaldehyde is used for cross linking (A) and Rapigest for reversal of crosslinking (B).
Fig 3.
A 12% SDS-PAGE gel showing different elution fractions after crosslinking by formaldehyde and affinity assay (A) and affinity assay after reversal of crosslinking by Rapigest (B). Lane1: broad range protein marker, lane 2–3: elution fractions. The proteins identified and sequence coverage corresponding to each band is shown.
Fig 4.
A 12% SDS-PAGE gel showing different elution fractions after crosslinking by formaldehyde and affinity assay.
(A) The affinity assay was performed using biotinylated primer specific to the dsz promoter attached to streptavidin coated dynabeads. The crosslinked crude extract was passed through the above column without digestion with T5 exonuclease. (B) The affinity assay was performed without attaching any oligonucleotide to streptavidin coated dynabeads. The crosslinked crude extract was digested with T5 exonuclease, then passed through the above column and the different fractions were collected. Lane1: broad range protein marker, lane 2: crude protein extract, lane 3: flow through, lane 4: elution fraction. The proteins identified and sequence coverage corresponding to each band is shown.
Fig 5.
A 12% SDS-PAGE gel showing different elution fractions after crosslinking by formaldehyde and affinity assay.
The affinity assay was performed using biotinylated primer of kanamycin promoter attached to streptavidin coated dynabeads. The crosslinked crude extract was digested with T5 exonuclease, then passed through the above column and the different fractions were collected. Lane1: broad range protein marker, lane 2: flow through, lane 3–4: elution fractions. The proteins identified and sequence coverage corresponding to each band is shown.
Fig 6.
Interactome of proteins obtained after affinity chromatography.
XRE family protein, WhiB, MerR family protein, Ubiquitin like protein pup, RNA polymerase -70 sigma factor, rpoZ (transcriptase subunit omega) and rbpA (RNA polymerase binding protein).
Fig 7.
A 12% SDS-PAGE gel showing overexpression of our protein in different expression strains.
Lane1: marker, lane2: BL21(DE3) uninduced, lane3: BL21(DE3) 5hr induction, lane4: BL21(DE3) overnight induction, lane5: BL21(DE3) pLysS uninduced, lane6: BL21(DE3) pLysS 5hr induction, lane7: BL21(DE3) pLysS overnight induction.
Fig 8.
Gel shift assay with partially purified XRE family transcription regulator.
Lane 1: free Cy5 labeled dsz, lane 2–5: Cy5 labeled dsz + partially purified XRE (1, 2, 3,4 μg respectively), lane 6: control lane (it contains other proteins of partial purified lane except XRE family protein), lane 7–10: Cy5 labeled dsz + partially purified XRE concentrated (5, 10, 15,20 μg respectively).