OPENING OF AN RNA-POLYMERASE-II PROMOTER OCCURS IN 2 DISTINCT STEPS AND REQUIRES THE BASAL TRANSCRIPTION FACTORS IIE AND IIH

We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that crea ting a region of heteroduplex DNA around the start site of the adenovi rus major late (AdML) promoter circumvents the requirement for TFIIE a nd TFIIH in transcription. The critical size and position of the heter oduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from -4 to +2. Promoter opening was investigated dire ctly with potassium permanganate (KMnO4), a chemical probe specific fo r single-stranded thymidines. We found that KMnO4-detectable opening o f the AdML promoter requires the presence of the complete pre-initiati on complex, DBpolFEH, and that opening occurs in two discrete steps. F irst, dependent on ATP but prior to initiation, the -9 to +1 region be comes single-stranded. Second, formation of the first phosphodiester b ond results in expansion of the open region to the +8 position. Our re sults lead to a model in which the critical function of the TFIIH-asso ciated DNA helicases is to create a single-stranded region. This gives RNA polymerase II access to the nucleotides of the template strand an d allows expansion of the open region upon formation of the first phos phodiester bond.