Interactions between repressor and anti-repressor elements in the carbamyl phosphate synthetase I promoter

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 binds a factor which is unrelated to the other upstream binding site factors. Sites I-III, on the other hand, bind the same or similar factors, but with different affinities (site II > site III > site I). High affinity binding to site II requires the core octamer, GTTGCAAC. Sequential 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 context of a -157 promoter in which the GTTGCAAC core of site II had been mutated, promoter activity was completely repressed, and this repression was due to sites I and III. Repression by sites I and III in the presence 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 synthetase 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 element in site II suggest that the anti-repressor may interfere with the site I and site III repressors by quenching.