Journal: Nucleic Acids Research
Article Title: VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae
Figure Lengend Snippet: Summary of in vitro primer extension and DNase I and KMnO 4 footprinting. ( A ) Sequence of P vpsL from −60 to +30. Bold and underlined A with black arrow at +1 and bold G (+3) represent the TSS determined by primer extensions; the −10 element and the −35 region are labeled and boxed in green; sequences in bold and red denote the VpsR binding site. Protection sites from DNase I footprinting and hypersensitive sites are depicted as rectangular boxes and triangles, respectively, either above (non-template) or below (template) the sequences: gray, RNAP with or without c-di-GMP or VpsR; black, RNAP with VpsR and c-di-GMP; red, VpsR with or without c-di-GMP. The open transcription bubble detected using KMnO 4 footprinting is shown as separated ssDNA from position −11 to +2 with sites of KMnO 4 cleavage indicated as purple asterisks. ( B ) Summary of positions of protection and hypersensitive sites on non-template and template strand DNA.
Article Snippet: Cells were harvested by centrifugation and RNA was extracted using the RNeasy kit (Qiagen), and an on-column DNase I digestion (Qiagen) was performed according to manufacturer's instructions.
Techniques: In Vitro, Footprinting, Sequencing, Labeling, Binding Assay