Now that we have the layout of how RNAPII works during transcription, we can look at what causes changes in the normal function of transcription with inhibitors such as α-amanitin. α-Amanitin is the most potent and specific known inhibitor of the RNAPII enzyme [1], [2]. α-Amanitin (Show structure) is produced by the poisonous mushroom Amanita phalloides which is also called the destroying angel. This is actually such a potent inhibitor to RNAPII that more than a hundred deaths result worldwide from the ingestion of the poisonous mushroom. Ingestion of the mushroom will usually kill humans by destroying the liver and damaging the kidneys [2]. Bushnell et al. showed that the binding site of α-amanitin is located beneath the bridge helix (Bridge Helix) in a funnel shaped cavity in the RNAPII structure known as pore 1. The RNAPII residues that interact with α-amanitin are primarily located on that bridge helix and in fact there is a strong hydrogen bond formed between the bridge helix and α-amanitin.
What actually happens is α-amanitin will penetrate pore 1 and regulate translocation and stop elongation at or before translocation occurs [2].The binding of α-amanitin does not affect NTP loading into the active site because it sits too far away from the active site [1], [2]. How it prevents translocation is that the bridge helix needs flex in order to allow DNA movement. Since α-amanitin is associating with the bridge helix, it prevents this flexing and thus prevents DNA translocation [1], [2]. Gong et al. showed that depending on when α-amanitin associates with RNAPII will determine how it inhibits the enzyme. At some points, they are allowed to form a single phosphodiester bond but the α-amanitin will inhibit the next bond addition cycle. Other studies have shown that α-amanitin can allow for multiple phosphodiester bonds to form but the elongation rates are highly reduced [2]. This could have happened by the hydrogen bond between α-amanitin and the bridge helix being broken which would have allowed for the translocation of the DNA, and the enzyme would become resistant through a single bond addition cycle before inhibition was restored [2].
[1] Bushnell, D.A.; Cramer, P.; Kornberg, R.D. Structural basis of transcription: α-Amanitin-RNA polymerase II cocrystal at 2.8 Å resolution. PNAS. 5 February 2002. 99, 1218-1222.
[2] Gong, X.Q.; Nedialkov, Y.A.; Burton, Z.F. α-Amanitin Blocks Translocation by Human RNA Polymerase II. The Journal of Biological Chemistry. 19 April, 2004. 279, 27422-27427.