Influence of substrate and template binding on the interaction of α-amanitin with yeast RNA polymerase II: fluorescence spectroscopic analysis

Abstract

Fluorescence titration of the tryptophan residues in yeast RNA polymerase II with a-amanitin shows there are two types of binding between the inhibitor and the enzyme. When the stronger binding site was saturated with a-amanitin, the enzyme-inhibitor complex could not be further titrated efficiently with ATP, indicating that probably the inhibitor and the substrate binding domains on the enzyme are overlapping. Supercoiled plasmid DNA bearing alcohol dehydrogenase I promoter of yeast showed binding with purified yeast RNA polymerase II in the absence of substrates. However, when the enzyme-inhibitor complex was titrated with this template, it was found that the complex behaves in a similar way as the enzyme alone towards the template DNA. It suggests that probably the inhibitor and the template binding sites on the enzyme are quite different.