Is. Goping et Gc. Shore, INTERACTIONS BETWEEN REPRESSOR AND ANTI-REPRESSOR ELEMENTS IN THE CARBAMYL-PHOSPHATE SYNTHETASE-I PROMOTER, The Journal of biological chemistry, 269(5), 1994, pp. 3891-3896
The proximal promoter of the rat carbamyl phosphate synthetase I gene
contains four cis-acting regulatory regions, designated sites GAG, I,
II, and III. The GAG site, which is located adjacent to the predicted
TATA region, contains a direct repeat of the nonamer, GAGGAGGGG, and b
inds a factor which is unrelated to the other upstream binding site fa
ctors. Sites I-III, on the other hand, bind the same or similar factor
s, but with different affinities (site II > site III > site I). High a
ffinity binding to site II requires the core octamer, GTTGCAAC. Sequen
tial 5'-deletion of the promoter revealed that GAG is the predominant
activating element in transient transfection analyses and, on its own,
can sustain activity of a -67 promoter fragment. However, in the cont
ext of a -157 promoter in which the GTTGCAAC core of site II had been
mutated, promoter activity was completely repressed, and this repressi
on was due to sites I and III. Repression by sites I and III in the pr
esence of mutated site II was relieved either by mutating sites I and
III or by introducing a 76-bp random DNA fragment between GAG and site
I. Our results suggest a model in which the carbamyl phosphate synthe
tase I promoter is controlled by a single activating element, GAG, and
by two repressor elements, sites I and III. We conclude that the high
affinity site II element binds an anti-repressor. Footprint analyses
of the -157 promoter with and without a mutation of the GTTGCAAC eleme
nt in site II suggest that the anti-repressor may interfere with the s
ite I and site III repressors by quenching.