RNA-POLYMERASE-II TRANSCRIPTION .2. RATE OF PROMOTER CLEARANCE IS ENHANCED BY A PURIFIED ACTIVATING TRANSCRIPTION FACTOR CAMP RESPONSE ELEMENT-BINDING PROTEIN

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/ cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus se quence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE- binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptio nal activator in vitro using a HeLa nuclear extract transcription syst em (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305) . In this study, we determined the mechanism of in vitro transcription al activation by this purified PBP. We used a PBP-depleted HeLa nuclea r extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-n ucleotide run-off product formation indicated that stimulation of tran scriptional activity by PBP was due entirely to an increase in the rat e constant for promoter clearance. Thus, under our conditions, the pur ified PBP had no effect on the rate of closed preinitiation complex fo rmation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide trans cript was stimulated by PBP. We found that the rate of closed preiniti ation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA poly merase transcription. The results also indicated that PBP binding to t he ATF/ CRE is required for the stimulation of promoter clearance. The se studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mam malian promoter.