GENE ACTIVATION BY THE ARAC PROTEIN CAN BE INHIBITED BY DNA LOOPING BETWEEN ARAC AND A LEXA REPRESSOR THAT INTERACTS WITH ARAC - POSSIBLE APPLICATIONS AS A 2-HYBRID SYSTEM

The Escherichia coli activator and repressor proteins AraC and LexA bi nd DNA as homodimers. Here we show that their heterodimerization throu gh fused cognate dimerization domains results in repression of AraC-de pendent gene activation by LexA. Repression also requires a LexA opera tor half-site located several helical turns downstream of the AraC ope rator. This requirement for a specific spatial organization of the ope rators suggests the formation of a DNA loop between operator-bound Ara /LexA heterodimers, and we propose that heterodimerization with the Ar aC hybrid provides co-operativity for operator binding and repression by the LexA hybrid. Consistent with a mechanism that involves DNA loop ing, repression increases when the E. coli DNA looping and transcripti onal effector protein IHF binds between the AraC and LexA operators. T hus, we have combined the functions of three distinct transcriptional effector proteins to achieve a new mode of gene regulation by DNA loop ing, in which the activator protein is an essential part of the repres sor complex. The flexibility of the DNA loop may facilitate this novel combinatorial arrangement of those proteins on the DNA, The requireme nt for protein interactions between the AraC and LexA hybrids for gene regulation suggests that this regulatory circuit may prove useful as an E. coli-based two-hybrid system.