BINDING OF THE TORR REGULATOR TO CIS-ACTING DIRECT REPEATS ACTIVATES TOR OPERON EXPRESSION

The expression of the Escherichia coli torCAD operon, which encodes th e anaerobically expressed trimethylamine N-oxide (TMAO) reductase resp iratory system, requires the presence of TMAO in the medium. The respo nse regulator, TorR, has recently been identified as the regulatory pr otein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a de cameric consensus motif designated the for boxes. Alteration by base s ubstitutions of any of the four for boxes in a plasmid containing a to rC'-lacZ fusion dramatically reduces TorR-dependent torC expression. I n addition, deletion of the distal for box (box1) abolishes torC induc tion whereas the presence of a DNA fragment starting three bases upstr eam from box1 suffices for normal torC expression. Footprinting and ge l-retardation experiments unambiguously demonstrated that TorR binds t o the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two for boxes (box1 and box2); the second is located upstream from the -3 5 promoter sequence and includes the third for box (box3); the last is found downstream from the -35 sequence and corresponds to the fourth for box (box4). Binding to the upstream for boxes (box1 and box2) appe ars to be stronger than binding to the downstream for boxes (box3 and box4) since only the upstream region is protected at the lower concent ration of TorR used in the footprinting experiments. We propose a mode l in which multiple binding sites (i.e. the for boxes) contribute to t he formation of a nucleoprotein complex, but only one particular proxi mal site positions TorR properly so that it interacts with RNA polymer ase.