Efficient control of raf gene expression by CAP and two Raf repressors that bend DNA in opposite directions

The plasmid-borne raf operon of Escherichia coli encodes proteins involved in the uptake and utilisation of the trisaccharide raffinose. The operon is subject to dual regulation; to negative control by the binding of RafR rep ressor to twin operators, O-1 and O-2, and to positive control by the cAMP- binding protein, CAP. We have identified the CAP binding site (CBS) as a 22 bp palindromic sequence with incomplete dyad symmetry by deletion analysis , DNaseI footprinting and electrophoretic mobility shift assays (EMSA) of C AP-DNA complexes. The CBS is centred 60.5 bp upstream of the transcription start point and partially overlaps O-1. In vivo, CAP increases rafA (alpha- galactosidase) gene expression up to 50-fold. The 28 bp spacing between the centres of CBS and the -35 box is essential, since insertions of 4, 8, 12 or 16 bp completely eliminated rafA gene expression. In vitro binding studi es revealed that the CBS, O-1 and O-2 sites, can be simultaneously occupied by their cognate proteins. However, no cooperativity between binding of CA P and RafR was detected. EMSA with circularly permuted DNA fragments demons trated that CAP and RafR proteins bend raf promoter (rafP) DMA by 75 degree s +/- 5 degrees and 95 degrees +/- 5 degrees, respectively, in opposite dir ections. Among sugar catabolic operons, the compact arrangement of three pr otein-binding sites, a CBS and two operators bounding the -35 promoter box, is unique and provides a sensitive and highly efficient device for transcr iptional control.