Action of α-amanitin during pyrophosphorolysis and elongation by RNA polymerase II

Abstract

Using defined elongation complexes formed on dC-tailed templates with Drosophila RNA polymerase II, we have examined elongation, pyrophosphorolysis, and DmS-II-mediated transcript cleavage and the inhibitory effect of α-amanitin on these processes. Analysis of pyrophosphorolysis on soluble or immobilized DNA templates confirmed that NTPs are liberated instead of dinucleotides that are released during DmS-II- meditated transcript cleavage. 10 μg/ml α-amanitin completely inhibited DmS-II-mediated transcript cleavage but allowed extended pyrophosphorolysis and nucleotide addition to occur. α-Amanitin dramatically decreased the V(max) for nucleotide addition but only slightly affected the K(m) for nucleotides. Although the processes are mechanistically distinct, both pyrophosphorolysis and DmS-II-mediated transcript cleavage frequently resulted in similar patterns of shortened transcript. Since polymerase molecules encounter similar kinetic barriers during both processes, it is possible that there is a common step in the reverse movement of the polymerase.