Having the layout of how RNAPII functions during transcription, scientists can look at what causes changes in the normal function of transcription with inhibitors such as alpha-amanitin. Alpha amanitin, is the product of the mushroom Amanita phalloides, or as it otherwise known as “the death cap”. This extremely toxic mushroom destroys the liver and kidney. A lethal dose for an adult is less than 3 grams, with symptoms developing 8-24 hours after ingestion.
The molecular target of a-amanitin is the inhibition of RNA polymerase II activity13. The binding site of a-amanitin was found to be beneath the “bridge helix” which extends across the cleft between the Rpb1 and Rpb2 subunits in the “funnel” cavity of pore 13. The hydroxyproline 2 region of a-amanitin hydrogen bonds with the bridge helix residue, along with the hydrogen binding of 4,5-dihydroxyisoleucine 3 with the bridge helix3. Contacts to the bridge helix are on the opposite face from the RNAP active site, meaning the a-amanitin isn’t likely blocking catalysis directly13. Amanitin is found to partially block the pore, used in NTP substrate loading and possibly pyrophosphate release, although it does not affect the NTP affinity for the RNAP II. Based on elongational stall studies, it was found that the pore that is blocked is a major allosteric site for regulation of transcriptional elongation, pausing, arrest, RNA cleavage, and transcriptional restart13.
Kinetic studies run have shown 3 conformational states when stalled, states A and B, pretranslocated, are susceptible to a-amanitin; however, state C, the posttranslocated form, is shown to be resistant to a-amanitin13. This is proven through the formation of multiple phosphodiester bonds that form without any dissociation of the inhibitor. Microcin J25 is a bacterial inhibitor of RNAP that is similar in structure and function. Microcin penetrates the pore of RNAP to inhibit initiation and elongation13. Both Microcin J25 and a-amanitin contain cyclic octapeptide structures, which obviously have a role in transcription inhibition.