Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites
| Autor: | Edward Tarien, Peter P. Chuknyisky, Richard B. Beal, Gunther L. Eichhorn, Joseph M. Rifkind |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Analytical chemistry
Biochemistry law.invention Metal chemistry.chemical_compound Adenosine Triphosphate Poly dA-dT law RNA polymerase Escherichia coli RNA polymerase I Molecule Electron paramagnetic resonance chemistry.chemical_classification Manganese Binding Sites biology Electron Spin Resonance Spectroscopy Temperature Active site DNA-Directed RNA Polymerases Zinc Crystallography Enzyme chemistry visual_art biology.protein visual_art.visual_art_medium Titration |
| Zdroj: | Biochemistry. 29:5987-5994 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00477a016 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|