DYNAMIC ASPECTS OF DNA-PROTEIN INTERACTIONS IN THE TRANSCRIPTIONAL INITIATION COMPLEX AND THE HORMONE-RESPONSIVE DOMAINS OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE PROMOTER IN-VIVO
S. Faber et al., DYNAMIC ASPECTS OF DNA-PROTEIN INTERACTIONS IN THE TRANSCRIPTIONAL INITIATION COMPLEX AND THE HORMONE-RESPONSIVE DOMAINS OF THE PHOSPHOENOLPYRUVATE CARBOXYKINASE PROMOTER IN-VIVO, The Journal of biological chemistry, 268(33), 1993, pp. 24976-24985
Transcription initiation of the gene encoding phosphoenolpyruvate carb
oxykinase (PEPCK) is stimulated by glucocorticoids and glucagon, via c
AMP, and dominantly inhibited by insulin in rat liver and H4IIE cells.
Lysolecithin-permeabilized H4IIE cells recover completely and continu
e to multiply, yet are transiently penetrable by macromolecules. These
cells, after various hormonal treatments, were utilized for in situ D
Nase I protection studies of the PEPCK promoter. Nearly all of the sit
es of protein interaction observed in vitro are protected in vivo as w
ell as several additional sites. The DNase I protection pattern is the
same in cells without or with any of the hormone treatments, suggesti
ng that hormonal modulation of transcription does not involve addition
or removal of factors from the hormone response elements of the promo
ter. We focused on the organization and stability of the transcription
initiation complex as well as the dynamic nature of distal promoter f
actors in their interaction with DNA. The transcription initiation com
plex was detected, and it appears to be coexistent 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, whi
ch suggests that the complex is not broken down and reformed with each
round of initiation by RNA polymerase II. Other factors bind to the P
EPCK 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/in
sulin-responsive region of the promoter functions through factors whic
h are involved in a rapid exchange, suggesting quite different modes o
f transcriptional regulation.