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, pyrophospho rolysis, and DmS-II-mediated transcript cleavage and the inhibitory ef fect of alpha-amanitin on these processes. Analysis of pyrophosphoroly sis on soluble or immobilized DNA templates confirmed that NTPs are li berated instead of dinucleotides that are released during DmS-II-medit ated transcript cleavage, 10 mu g/ml alpha-amanitin completely inhibit ed DmS-II-mediated transcript cleavage but allowed extended pyrophosph orolysis and nucleotide addition to occur. alpha-Amanitin dramatically decreased the V-max for nucleotide addition but only slightly affecte d the K-m for nucleotides. Although the processes are mechanistically distinct, both pyrophosphorolysis and DmS-II-mediated transcript cleav age frequently resulted in similar patterns of shortened transcript. S ince polymerase molecules encounter similar kinetic barriers during bo th processes, it is possible that there is a common step in the revers e movement of the polymerase.