Electron Microscopic Localization of the Binding Sites of Escherichia coli RNA Polymerase in the Early Promoter Region of T7 DNA

Escherichia coli RNA polymerase bound to T7 DNA has been studied in the electron microscope. By dark-field observation of specimens prepared by adhesion on positively charged films, we have located the binding sites of RNA polymerase on the physical map of T7 DNA. At a molar ratio of RNA polymerase to DNA smaller than or equal to 10, we found three strong binding sites at 1.15, 1.41 and 1.65 map units from the left DNA end. At higher ratios additional binding sites were occupied. Two of them were located at 0.6 and 3.3 map units from the left DNA end. Preliminary results are presented on the binding of RNA polymerase to T7 DNA in the presence of rifampicin, heparin, actinomycin D and ethidium bromide. Only heparin added to the enzyme and ethidium bromide added to the DNA template inhibited the formation of the binding complexes. It is suggested that the three strong binding sites correspond to the three strong initiation sites found by other workers in the early promoter region of T7 DNA.