Structural studies on the active site of Escherichia coli RNA polymerase. 1. Interaction of metals on the i and i + 1 sites

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.     

Physiochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase.

In a medium containing 10mM Tris, pH 8, 10 mM MG++, 50 mM K+ and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 240 degrees at 37 degrees C. In this medium the total unwinding of the DNA increases linearly with the molar ratio of polymerase to DNA. The number of binding sites at which unwinding can occur is very large. If the K+ concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 140 degrees at 37 degrees C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA is strongly temperature dependent, and decreases with decreasing temperature.