HYDROXYL RADICAL FOOTPRINTS AND HALF-SITE ARRANGEMENTS OF BINDING-SITES FOR THE CYSB TRANSCRIPTIONAL ACTIVATOR OF SALMONELLA-TYPHIMURIUM

CysB is a transcriptional activator for the cysteine regulon and negat ively autoregulates its own gene, cysB. Transcription activation also requires an inducer, N-acetyl-L-serine. CysB is known to bind to activ ation sites just upstream of the -35 regions of the positively regulat ed cysJIH, cysK, and cysP promoters and to a repressor site centered a t about +1 in the cysB promoter. Additional accessory sites have been found in positively regulated promoters. The hydroxyl radical footprin ting experiments reported here indicate that the activation sites CBS- J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are com posed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp. N-Acetyl-L-serine stimulates binding to these sites as well as t o the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites. A second topology is found in the acc essory site CBS-K2 and the repressor site CBS-B, which contain diverge ntly oriented 19-bp half-sites separated by one or two helical turns. N-Acetyl-L-serine inhibits binding to these two sites. A third topolog y is present in the cysK and cysP promoters, where an additional half- site is oriented toward the activation site and separated from it by o ne helical turn. Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending. The marke d dissimilarities of these half-site arrangements and of their respons es to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to th em with different combinations of subunits.