The RNA polymerase II preinitiation complex formed in the presence of ATP

ATP hydrolysis is required for transcriptional initiation by RNA polymerase II in vitro. Reconstituted transcription using purified initiation factors and RNA polymerase II have revealed that the step dependent on ATP hydrolysis occurs at the same time as initiation of RNA synthesis. We report here that ATP hydrolysis is also required for formation of the preinitiation complex in crude extracts. Two distinct preinitiation complexes were identified, one formed in the presence and the other in the absence of ATP. These complexes were isolated by glycerol gradient centrifugation. The preinitiation complex formed in the presence of ATP was able to synthesize transcripts with addition of only ribonucleotide triphosphates, whereas the preinitiation complex formed in the absence of ATP was inactive and required addition of protein fractions and ATP. These results suggest that the inactive preinitiation complex is activated by addition of the protein fractions and ATP hydrolysis. The active preinitiation complex sedimented at approximately 40 S in glycerol gradient centrifugation, a rate similar to that of RNA polymerase II holoenzyme reported by Maldonado et al. [ Nature (1996), 381, 86-89].