Efficient Control of raf Gene Expression by CAP and Two Raf Repressors that Bend DNA in Opposite Directions

Abstract

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 repressor to twin operators, O₁ and O₂, 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 CAP-DNA complexes. The CBS is centred 60.5 bp upstream of the transcription start point and partially overlaps O₁. In vivo, CAP increases rafA (α-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 studies revealed that the CBS, O₁ and O₂ sites, can be simultaneously occupied by their cognate proteins. However, no cooperativity between binding of CAP and RafR was detected. EMSA with circularly permuted DNA fragments demonstrated that CAP and RafR proteins bend raf promoter (rafP) DNA by 75° ± 5° and 95° ± 5°, respectively, in opposite directions. Among sugar catabolic operons, the compact arrangement of three protein-binding sites, a CBS and two operators bounding the—35 promoter box, is unique and provides a sensitive and highly efficient device for transcriptional control.