ON THE ROLE OF THE ESCHERICHIA-COLI INTEGRATION HOST FACTOR (IHF) IN REPRESSION AT A DISTANCE OF THE PYRIMIDINE SPECIFIC PROMOTER P1 OF THECARAB OPERON

Binding of integration host factor to its target site, centered around nucleotide -305 upstream of the transcription startpoint, exerts anta gonistic effects on the expression of P1, the upstream pyrimidine spec ific promoter of the E coli and S typhimurium carAB operons. IHF stimu lates P1 promoter activity in minimal medium, but also increases the r epressibility of this promoter by pyrimidines. We present evidence str ongly suggesting that IHF exerts these effects by modulating the bindi ng of another pyrimidine specific regulatory molecule, probably the pr oduct of gene carP. The carAB control region contains a GATC Dam methy lation site, 106 bp upstream of the P1 transcription startpoint, which can be protected in vivo against methylation. This protection require s at least the regulatory carP gene product and a high pyrimidine nucl eotide pool and, as shown here, the integration host factor. Whether C arP directly binds to this site or exerts its protective effect indire ctly is not yet known. In the absence of IHF (himA) or in mutants affe cted in the IHF target site this protection is strongly impaired, sugg esting that IHF positively influences the formation or the stability o f the protective protein-DNA complex some 200 bp downstream. Furthermo re, we have demonstrated that the distance separating the IHF and GATC Dam methylase target sites is crucial for the in vivo protection and for pyrimidine mediated regulation of P1 promoter expression. Indeed, shortening this distance by 6 bp, and more surprisingly also by 11 bp, results in a severe reduction of the degree of in vivo protection of the GATC site against methylation and concomitantly of the repressibil ity by pyrimidines of P1 promoter activity. The absence of both these effects in a double, deletion-duplication, mutant resulting in a net i ncrease of the intervening sequence by 1 bp, clearly demonstrates that these effects are not due to the disruption of an important regulator y site, but must be attributed to variations in the distance separatin g different protein binding sites.