MULTIPLE ATP-DEPENDENT STEPS IN RNA-POLYMERASE-II PROMOTER MELTING AND INITIATION

Permanganate probing and abortive initiation assays were used to inves tigate the role of ATP in several successive stages of transcription i nitiation at the activated adeno E4 and mouse DHFR promoters. Removal of ATP at several points along the multi-step pathway blocked further progress towards its completion. Most strikingly, even if the DNA tran scription start site is opened using ATP, the subsequent removal of AT P disallows formation of the first phosphodiester bond of the RNA. Aft er ATP-dependent formation of a short RNA, a new transcription complex forms, which is more stable and has a longer open region. Both RNA an d ATP appear to play roles in the formation of this complex, The need for ATP throughout this multistep initiation pathway leads to new and unexpected possibilities for the use of energy and ATPases in transcri ption initiation.