Journal: Nature Communications
Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions
Figure Lengend Snippet: In vitro transcription assays to measure RNA release. a Cartoon showing the DNA template used for the study. Promoter and the terminator regions are indicated. T–T dimers, generated from the UV-exposure, formed at various sites on the template strands are indicated by (*). ECs with variable RNA chain length are shown to get stalled at the lesions that are located at the proximal most sites from the transcription stat-site. Mfd or Rho dislodges these stalled ECs and release RNA molecules of variable chain length that are analyzed by gel-electrophoresis. Rho loads onto these RNA once the latter reaches the critical lengths of 60–90 nt. Rho and Mfd proteins are indicated. b Autoradiogram showing selective degradation of UV-irradiated DNA by T4-PDG. Radioactive labeled transcription template DNA, with or without UV exposure, was subjected to the cleavage with T4-PDG. c and d Autoradiograms showing in vitro transcription performed on the immobilized DNA templates under different conditions as indicated. S denotes half of the RNA release in the supernatant and P denotes the other half of RNA and the total pellet. Transcripts those reached at the end of the template are denoted as run-off (RO). Zones of RNA release are shown next to the autoradiograms by dashed lines. Fractions of RNA release is calculated as: 2S/([S]+[S+P]). Amounts of RNAP, DNA, Rho, and Mfd were 25, 10, 100, and 100 nM, respectively
Article Snippet: Formation of T–T dimer were determined from the sensitivity of the UV-exposed template to the enzyme T4-PDG (NEB, USA).
Techniques: In Vitro, Generated, Nucleic Acid Electrophoresis, Irradiation, Labeling