Transcription initiation by RNA polymerase II in vitro. At least two nucleotides must be added to form a stable...

We have prepared RNA polymerase II preinitiation complexes by incubating templates containing the adenovirus 2 major late promoter with HeLa cell nuclear extracts in the absence of nucleoside triphosphates. These preinitiation complexes are partially purified by gel filtration and are then provided with the appropriate substrates to allow either one or two phosphodiester bonds to be formed. When substrates that allow only one bond to form are used, no stable ternary complex is obtained and no RNA is made that can be incorporated into longer RNA chains. A stable complex is obtained, however, if the RNA polymerase can make two bonds. The production of a stable ternary complex requires ATP or dATP and is inhibited by alpha-amanitin. In the course of exploring the energy requirement for initiation we found that dATP may be incorporated, in the absence of ATP, as the initial base of the RNA. However, deoxyribonucleotides are not appreciably incorporated into the body of the transcript after the first two bases have been added to the growing chain.