Interaction of T7 RNA polymerase with affinity modifier analogs of nucleoside triphosphates

To study the functional topography of T7 RNA polymerase and the localization of sites of the enzyme contacts with the template and the RNA product, a series of nucleotide analogs containing reactive groups potentially capable of covalent interaction with spatially close amino acid residues of the protein were investigated. The UTP analogs exhibited pronounced properties as T7 RNA polymerase substrates, which permitted identification of RNA products containing these modified nucleotides. The kinetics of incorporation of these derivatives into RNA was studied, the corresponding constants were determined, and the dependence of the reaction mechanism on the type of nucleotide modification was demonstrated. The inhibitory effect of the initiating nucleotide derivative 6-thioGTP on T7 RNA polymerase was shown. This effect was markedly enhanced after UV irradiation, which was indicative of irreversible binding of the derivative to the enzyme. The derivatives of the consensus T7 promoter with modified nucleotides in different positions were obtained, their catalytic competence and applicability as highly specific affinity reagents were investigated.