Differential Fluorescence and Kinetic Studies on the Template-Binding of RNA Polymerase from Parsley and Escherichia coli

Abstract

A fluorescence spectroscopy method is described for studying association of RNA polymerase with DNA templates. Using double beam differential fluorescence at excitation and emission wavelengths of 285 and 335 nm, respectively, the new technique discriminates non-specific decrease of fluorescence intensity by addition of DNA from quenching of polymerase fluorescence by protein-nucleic acid interactions. Comparing the results with studies of UMP incorporation into RNA, the Ks-values of template-binding were in good agreement with the values for RNA synthesis, pointing to specific interaction of polymerase and the DNA template measured by the fluorescence method. While E. coli enzyme showed higher affinity for templates such as heat- denatured poly [d(A-T)] and poly [d(G-C)] parsley RNA polymerase I accepted such templates with the same affinity as salmon sperm DNA. It is obvious that divalent cations are not necessary for the interaction of both enzymes with single-stranded DNA-templates. UMP incorporation studies suggest that transcription is a cooperative process.