A soluble biotinyl-α-amanitin affinity probe for the isolation of RNA polymerase B

Abstract

Utilizing the 6′-hydroxyindole moiety of α-amanitin for substitution, biotinyl-α-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-α-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 × 10−8 M as compared with a Ki of 5 × 10−9 M for natural α-amanitin. RNA polymerase B complexed with biotinyl-α-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the amatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-α-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.