Effect of Sarkosyl and heparin on single-step addition reactions catalysed by wheat-germ RNA polymerase II–poly[d(A–T)]transcription complexes

Abstract

Incubation of purified wheat-germ RNA polymerase II with poly[d(A-T)] template, Mn2+, U-A dinucleoside monophosphate primer and UTP substrate resulted in catalytic formation of the trinucleoside diphosphate U-A-U, in accordance with the results of previous studies. Both Sarkosyl and heparin inhibited completely and immediately (within less than 1 min) U-A-U synthesis, if either of these compounds was added to the assays during the progress of the reaction. This behaviour is in marked contrast to that reported for single-step addition reactions catalysed by Escherichia coli RNA polymerase on the same template [Sylvester & Cashel (1980) Biochemistry 19, 1069-1074]. However, treatment of the transcription complexes with Sarkosyl or heparin for periods sufficient to abolish U-A-U formation completely did not suppress completely the ability of such complexes to elongate RNA chains. Hence, the effect of Sarkosyl or heparin on the rate of U-A-U synthesis was predominantly due to change in the rate (or in the mechanism) of trinucleotide product release by the transcription complexes. Furthermore, once U-A-U synthesis has begun on the poly[d(A-T)] template, the transcription complexes became resistant to the action of a competitor DNA such as poly[d(G-C)]. The results are consistent with a model where at least a sizeable fraction of the enzyme molecules remains associated with the DNA template upon formation of a single phosphodiester bond.