Interactions between RNA polymerase II, factors, and template leading to accurate transcription

Accurate transcription by RNA polymerase II has been shown to require multiple factors in addition to the purified polymerase. In this study, we use a reconstituted transcription system, consisting of purified RNA polymerase II and three essential HeLa cell chromatographic fractions, to study events leading to transcription from the adenovirus major late promoter. A preincubation-pulse-chase protocol resolves the reaction into events occurring before and after nucleotide addition. Preincubation of template with a mixture of RNA polymerase II and factors allows formation of "activated" complexes, which are defined by the ability to rapidly commence accurate transcription when presented nucleotides. Maximal activation requires that polymerase, template, and each of the three HeLa fractions be present during preincubation. The activated complexes are template associated, as shown by their inability to exchange onto a second template added during further preincubation. Similar protocols are used to define functional intermediates leading to the activated complex. A template-associated functional complex is formed during the preincubation of template with just two of the HeLa fractions. Polymerase can associate with this intermediate complex in the absence of the third HeLa fraction. In the accompanying paper, we describe a direct analysis of initiation by "activated" complexes.