Highly selective affinity labelling of E. coli RNA polymerase promoter complex with reactive derivatives of oligonucleotide primers of various chemical specificities
I G. Tsarev, A. A. Mustaev, E. F. Zaychikov, T. Yu. Alikina, A. G. Venyaminova, M. N. Repkova
Immunological Institute, Siberian Division, Academy of Sciences of the USSR, Irkutsk: Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Academy of Sciences of the USSR
Abstract: A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligo-nucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labeling. The procedure resulted in labeling DNA and β-, β’- or σ-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labeling patterns as a function of the oligonuclcotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.
Russian Journal of Bioorganic Chemistry 1990, 16 (6):765-779