IN-VIVO DEGRADATION OF RNA-POLYMERASE-II LARGEST SUBUNIT TRIGGERED BYALPHA-AMANITIN

alpha-Amanitin is a well-known specific inhibitor of RNA polymerase II (RNAPII) in vitro and in vivo, It is a cyclic octapeptide which binds with high affinity to the largest subunit of RNAPII, RPB1, We have fo und that in murine fibroblasts exposure to alpha-amanitin triggered de gradation of the RPB1 subunit, while other RNAPII subunits, RPB5 and R PB8, remained almost unaffected, Transcriptional inhibition in alpha-a manitin-treated cells was slow and closely followed the disappearance of RPB1, The degradation rate of RPB1 was alpha-amanitin dose dependen t and was not a consequence of transcriptional arrest, alpha-Amanitin- promoted degradation of RPB1 was prevented in cells exposed to actinom ycin D, another transcriptional inhibitor, Epitope-tagged recombinant human RPB1 subunits were expressed in mouse fibroblasts, In cells expo sed to alpha-amanitin the wild-type recombinant subunit was degraded l ike the endogenous protein, but a mutated alpha-amanitin-resistant sub unit remained unaffected, Hence, alpha-amanitin did not activate a pro teolytic system, but instead its binding to mRPB1 likely represented a signal for degradation, Thus, in contrast to other inhibitors, such a s actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, w hich reversibly act on transcription, inhibition by alpha-amanitin can not be but an irreversible process because of the destruction of RNAPI I.