Structural studies on the active site of Escherichia coli RNA polymerase. 2. Geometrical relationship of the interacting substrates

• Published in: Biochemistry (Easton) Vol. 29; no. 25; pp. 5994 - 6002
• Main Authors: Beal, Richard B, Pillai, Rajasekharan P, Chuknyisky, Peter P, Levy, Abraham, Tarien, Edward, Eichhorn, Gunther L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.06.1990
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacology; Binding Sites; DNA-Directed RNA Polymerases - metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli; Escherichia coli - enzymology; Magnetic Resonance Spectroscopy - methods; Manganese - pharmacology; Models, Chemical; Poly dA-dT - metabolism; Protein Conformation; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00477a017

Since a major function of RNA polymerase must be to bring together substrates in the optimal configuration for internucleotide bond formation, studies have been undertaken to understand the geometrical relationship of the two substrates. A model has been constructed for the geometry of interaction of two ATP molecules poised on the active site of the Escherichia coli enzyme for the formation of the first bond in RNA synthesis. The model is based primarily on the distance, measured by EPR, between the two metals in the i and i + 1 subsites, as well as distances, measured by NMR, from each metal to points on the substrate in the same subsite, in the presence of a poly(dAdT).poly(dAdT) template. Both the Zn(II) in the i site and the Mg(II) in i + 1 are displaced by Mn(II). The nucleotide bases are not parallel to each other, in line with the reaction of the ATP molecules with DNA within the transcription bubble. The metal in the i site appears too far removed from substrate to participate in catalysis, but the metal in i + 1 is in position to bind to the beta- and gamma-phosphate groups and probably is involved in cleavage of the triphosphate, as has been previously suggested.