ABSTRACT
A frequently used schematic model of transcriptional elongation shows an RNA polymerase molecule moving along a linear DNA. This model is of course highly idealized and not compatible with promoter sequences [Gralla, J. D. (1991) Cell 66, 415-418; Schleif, R. (1992) Annu. Rev. Biochem. 61, 199-223] and regulatory proteins [Koleske, A. J., and Young, R. A. (1995) Trends Biochem. Sci. 20, 113-116; Dunaway, M., and Dröge, P. (1989) Nature 341, 657-659; Müller, H. P., Sogo, J. M., and Schaffner, W. (1989) Cell 58, 767-777] located some distance away from the point of transcription initiation [Karsten, R., von Hippel, P. H., and Langowski, J. (1995) Trends Biochem. Sci. 20, 500-506]. These circumstances lead to the expectation of curvature along the DNA strand and require looping between sometimes distant points. We have now shown curvature in a dinucleotide formed at the very onset of transcription when it is poised for reaction with a mononucleotide to form a trinucleotide. The curvature became evident from the demonstration that a metal ion bound with a mononucleotide in the i+1 (elongation) site is approximately equidistant from bases at the 5' end (i-1 site) and 3' end (i site) of the dinucleotide. Similar results were obtained with three different dinucleotides and four mononucleotides. Curvature of the RNA initiate may reflect curvature of the DNA to which it is bound. These studies show curvature to be a significant feature in the interaction between DNA template and RNA elongate even at the very beginning of transcription.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , Dinucleoside Phosphates/chemistry , Escherichia coli/enzymology , Nucleic Acid Conformation , Transcription, Genetic , Adenosine Triphosphate/chemistry , Binding Sites , DNA-Directed RNA Polymerases/genetics , Dinucleoside Phosphates/genetics , Magnetic Resonance Spectroscopy , Manganese/chemistry , Protons , Templates, GeneticABSTRACT
Distances between the metal ions bound to the product terminus i site and the substrate i + 1 site of Escherichia coli RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship. The metal bound to the substrate at the i + 1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the distance to base and ribose protons changes. The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position. In this scheme a metal-triphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Models, Structural , Nucleotides/metabolism , Transcription, Genetic , Base Composition , Binding Sites , Metals/metabolism , Models, Molecular , Molecular ConformationABSTRACT
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.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Escherichia coli/enzymology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Binding Sites , Electron Spin Resonance Spectroscopy , Magnetic Resonance Spectroscopy/methods , Manganese/pharmacology , Models, Chemical , Poly dA-dT/metabolism , Protein Conformation , TemperatureABSTRACT
The two substrates between which an internucleotide bond is formed in RNA synthesis occupy two subsites, i and i + 1, on the active site of Escherichia coli RNA polymerase, and each subsite is associated with a metal ion. These ions are therefore useful as probes of substrate interaction during RNA synthesis. We have studied interactions between the metals by EPR spectroscopy. The Zn(II) in the i site and the Mg(II) in the i + 1 site were substituted separately or jointly by Mn(II). The proximity of the metals was established by EPR monitoring of the titration at 5.5 K of the enzyme containing Mn(II) in i with Mn(II) going into the i + 1 site, and the 1:1 ratio of the metals in the two sites was confirmed in this way. The distance between the two metals was determined by EPR titration at room temperature of both the enzyme containing Zn(II) in i and Mn(II) in i with Mn(II) going into the i + 1 site, making use of the fact that EPR spectra are affected by dipolar interactions between the metals. The distances calculated in the presence of enzyme alone, in the presence of enzyme and two ATP substrates, and when poly(dAdT).poly(dAdT) was added to the latter system ranged from 5.2 to 6.7 A.
