title: Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.
abstract: T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.
authors.affiliation: Biophysics Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700064, India.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Pai
authors.forename: Mili
authors.initials: M
authors.lastname: Das
authors.forename: Rahul
authors.initials: R
authors.lastname: Banerjee
authors.forename: Dipak
authors.initials: D
authors.lastname: Dasgupta
mesh_terms: D001665:Binding Sites; D004396:Coloring Agents / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D014227:Triazines / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004396:Coloring Agents; D009711:Nucleotides; D014227:Triazines; D014764:Viral Proteins; C012842:Cibacron Blue F 3GA; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases