Photoreactivation of amanitin-inhibited RNA polymerase II.

The fungal toxin alpha-amanitin binds very tightly to RNA polymerase II (Kd approximately 10(-9) M) and inhibits the polymerizing activity of the enzyme. However, it has been unclear from previous studies whether or not this inhibition can be reversed. We show in this communication that it is possible to reverse the amanitin inhibition of polymerase by using a novel photoreactivation technique. When the inactive amanitin-wheat germ polymerase II complex is exposed to ultraviolet irradiation of a wavelength which is absorbed by the amanitin but not by the polymerase (monochromatic 314-nm irradiation), the enzyme recovers virtually all of the activity that an uninhibited control exhibits. Ultraviolet irradiation of a wavelength which is not absorbed significantly by the amanitin (monochromatic 350-nm irradiation) does not reactivate the inhibited polymerase. This ability to photoreactivate polymerase is discussed with respect to the mechanism of action of amanitin inhibition. Also discussed is the use of this technique for photoelution of an amanitin-Sepharose affinity column which we are developing for the isolation of transcriptionally active chromatin.