Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli

Efficient repression of the two promoters P1 and P2 of the gal operon requi res the formation of a DNA loop encompassing the promoters, In vitro, DNA l ooping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (O-E and O-I) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between O-E and O-I, and super coiled DNA, Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2, We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusi on of a reporter gene, gusA or lacZ, to each promoter individually, While t he P1 promoter was found to be repressible in the absence of DNA supercoili ng and HU, the repression of P2 was entirely dependent upon DNA supercoilin g in vivo. The P2 promoter was fully derepressed when supercoiling was inhi bited by the addition of coumermycin in cells, P2, but not P1, was also tot ally derepressed by the absence of HU or the O-I operator, From these resul ts, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing Gai n, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to O-E. The specific nucleoprotein complex es involving histone-like proteins, which repress promoter activity while r emaining sensitive to inducing signals, as discussed, may occur more genera lly in bacterial nucleoids.