Amanatin Toxin
As with any enzyme, the activity of RNA polymerase II can be stimulated or inhibited by various factors within a cell. One of the most potent and specific inhibitors of RNA polymerase II is alpha-amanatin, which comes from the poisonous Amanita Phalloides mushroom (1). The overall effect of amanatin on the transcription process is the inhibition of both initiation and elongation, thereby preventing the RNA from being transcribed, and furthermore, the protein encoded in the transcribed RNA molecule.
The action of amanitin directly affects the conformational change of RNA polymerase II that is necessary for the transcription mechanism (1). In the previous section, the structure of RNA polymerase II was discussed in detail, and throughout this section, I will be referring to that structure. The binding site of alpha-amanatin on the RNA polymerase II molecule is beneath a bridge helix and extends across the cleft between the two largest subunits, Rpb1 and Rpb2. The alpha-amanatin forms many hydrogen bonds with residues of the bridge helix and also with residues of the Rpb1 next to the bridge helix (1).
The interactions of α-amanatin with the polymerase II molecule imply that its mode of inhibition is not on the affinity of polymerase II to ribonucleotides, but rather it affects the translocation of RNA polymerase II along the DNA-RNA helix (1). Research has shown that addition of α-amanatin does not affect the formation of phosphodiester bonds. However, the rate at which the polymerase shifts along the hybrid helix is reduced to only a few nucleotides per minute in the presence of α-amanatin, compared to thousands per minute when it is absent (1). Consistent with these research findings is the physical location of the binding site of alpha-amanatin. It is physically too far from the active site to interfere with ribonucleotide entry and RNA synthesis (1).
Additional research and experiments involving derivatives of alpha-amanatin, such as proamanullin, also confirm these results. Proamanullin is similar in structure to alpha-amanatin, however, it lacks hydroxyl groups that form hydrogen bonds with RNA polymerase II (1). Results of the experiments revealed that proamanullin was 20,000 times less inhibitory that alpha-amanatin (1). Based on the research found through these experiments, alpha-amanatin allows the ribonucleotide entry into the active site but inhibits transcription by limiting the translocation of RNA polymerase II along the DNA-RNA double helix. By limiting this translocation, the active site never becomes empty and RNA synthesis ceases (1).