Amanitin Greatly Reduces the Rate of Transcription by RNA Polymerase II Ternary Complexes but Fails to Inhibit Some Transcript Cleavage Modes

Abstract

The toxin alpha-amanitin is frequently employed to completely block RNA synthesis by RNA polymerase II. However, we find that polymerase II ternary transcription complexes stalled by the absence of NTPs resume RNA synthesis when NTPs and amanitin are added. Chain elongation with amanitin can continue for hours at approximately 1% of the normal rate. Amanitin also greatly slows pyrophosphorolysis by elongation-competent complexes. Complexes which are arrested (that is, which have paused in transcription for long periods in the presence of excess NTPs) are essentially incapable of resuming transcription in the presence of alpha-amanitin. Complexes traversing sequences that can provoke arrest are much more likely to stop transcription in the presence of the toxin. The substitution of IMP for GMP at the 3' end of the nascent RNA greatly increases the sensitivity of stalled transcription complexes to amanitin. Neither arrested nor stalled complexes display detectable SII-mediated transcript cleavage following amanitin treatment. However, arrested complexes possess a low level, intrinsic transcript cleavage activity which is completely amanitin-resistant; furthermore, pyrophosphorolytic transcript cleavage in arrested complexes is not affected by amanitin.