Successive binding of raf repressor to adjacent raf operator sites in vitro

The raf repressor negatively regulates the transcription of the raf operon which encodes functions required for the uptake and hydrolysis of raffinose in Escherichia coli. Overexpression of the repressor gene under lac promoter control led to the formation of inclusion bodies. These were partially purified by centrifugation, solubilized in 0.1% SDS and reactivated by dilution. DNase I protection and gel retardation experiments demonstrated the specific binding of raf repressor to DNA fragments that contained the previously identified raf operator, an element comprising two 18 bp palindromic nucleotide sequences that flank the -35 raf promoter box. By using DNA fragments with one, two, or four copies of the 18 bp palindrome, these experiments revealed concentration dependent, successive occupation of all available binding sites by raf repressor. Melibiose released the repressor from the operator complexes, whereas raffinose and other alpha-galactosides did not, indicating that melibiose is the actual inducer in vivo. We suggest that successive occupation by repressor of two strategically located operator sites is a specific type of stepwise down-regulation of gene expression in response to repressor concentration.