Dynamic aspects of DNA/protein interactions in the transcriptional initiation complex and the hormone-responsive domains of the phosphoenolpyruvate carboxykinase promoter in vivo.

Abstract

Transcription initiation of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via cAMP, and dominantly inhibited by insulin in rat liver and H4IIE cells. Lysolecithin-permeabilized H4IIE cells recover completely and continue to multiply, yet are transiently penetrable by macromolecules. These cells, after various hormonal treatments, were utilized for in situ DNase I protection studies of the PEPCK promoter. Nearly all of the sites of protein interaction observed in vitro are protected in vivo as well as several additional sites. The DNase I protection pattern is the same in cells without or with any of the hormone treatments, suggesting that hormonal modulation of transcription does not involve addition or removal of factors from the hormone response elements of the promoter. We focused on the organization and stability of the transcription initiation complex as well as the dynamic nature of distal promoter factors in their interaction with DNA. The transcription initiation complex was detected, and it appears to be co-existent with a short region of naked single-stranded DNA over the TATA box on the template strand, as determined by potassium permanganate reactivity. This complex is quite stable, even under conditions of much reduced RNA synthesis, which suggests that the complex is not broken down and reformed with each round of initiation by RNA polymerase II. Other factors bind to the PEPCK promoter with half-lives ranging from a few minutes to more than 40 min. The cAMP response element apparently involves transcriptional modulation achieved through modification of a bound factor (presumably cAMP response element-binding protein), whereas the glucocorticoid/insulin-responsive region of the promoter functions through factors which are involved in a rapid exchange, suggesting quite different modes of transcriptional regulation.