Abstract
Transcription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20-50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes.
Highlights
RNA polymerase II associates with promoters via multiprotein complexes and is subsequently released from these complexes to carry out transcript elongation
One very important class of activators fits into this uncertain category, the activating transcription factor cyclic AMP response element binding (ATF/CREB) protein family
We have studied activation of in vitro transcription via interactions at a cyclic AMP response element by the ATF/CREB family of factors
Summary
RNA polymerase II associates with promoters via multiprotein complexes and is subsequently released from these complexes to carry out transcript elongation. The contact between activator and TFIID apparently occurs whether or not the protein is phosphorylated [18, 19, 23, 24, 27] On this basis the ATF/CREB members have been suggested to be involved in recruitment of the GTFs and the polymerase. Other studies, conducted in HeLa extracts containing a wide range of factors, have suggested that activation in such extracts occurs instead at the step of promoter clearance [20, 21]. In these studies ATF/CREB was not required to form preinitiation complexes in which the DNA start site was melted. The protein isoform that bound the CRE and accomplished the activation in nuclear extract was reported to be unphosphorylated CREB-1 [28]
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