Spectroscopic studies on the mode of binding of ATP, UTP and α-amanitin with yeast RNA polymerase II

Abstract

The binding affinity between the substrates ATP and UTP with the purified yeast RNA polymerase II have been studied here in the presence and absence of Mn 2+. In the absence of template DNA, both ATP and UTP showed tight binding with the enzyme without preference for any specific nucleotide, unlike Escherichia coli RNA polymerase. Fluorescence titration of the tryptophan emission of the enzyme by nucleoside triphosphate substrates gave an estimated K d value around 65 μM in the absence of Mn 2+ whereas in the presence of Mn 2+, the K d was 20 μM. The effect of substrates on the longitudinal relaxation of the HDO proton in enzyme-substrate complex also yielded a similar K d value.