RNA polymerase: correlation between transcript length, abortive product synthesis, and formation of a stable ternary...

In order to investigate the relationship between the stability of the ternary complex RNA polymerase-T7 D111 DNA-RNA product and the length of the bound RNA product, we have developed a protocol for the production of stable ternary complexes of known length and composition. The assembly of the ternary complex is achieved by utilizing a dinucleotide tetraphosphate (pppApU) as a selective primer, which is augmented by one or more appropriate nucleotides. The labeled products were characterized by autoradiography of gel electrophoresis patterns, which were then quantified. The criterion for stability is the protection from perturbations (a salt-jump or a rifampicin challenge), which effectively inhibit initiation. The formation of a bound ribotetranucleotide ternary complex confers stability and terminates abortive product synthesis.