ACTION OF ALPHA-AMANITIN DURING PYROPHOSPHOROLYSIS AND ELONGATION BY RNA-POLYMERASE-II

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 alpha-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 mu g/ml alpha-amanitin completely inhibited DmS-II-mediated transcript cleavage but allowed extended pyrophosphorolysis and nucleotide addition to occur. alpha-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.