Purification and properties of RhaR, the positive regulator of the L-rhamnose operons of Escherichia coli.
The product of the rhaR gene, which regulates the level of mRNA produced from the four L-rhamnose-inducible promoters of the rhamnose operon, has been hypersynthesized and purified by a two-column procedure. The purified protein is a 33 kDa DNA-binding protein that binds to an inverted repeat structure located within the psr promoter, the promoter for the rhaS and rhaR genes. The equilibrium binding constants and kinetic constants have been determined under a variety of solution conditions. The protein binds with high affinity and its binding is sensitive to salt concentration and the presence of L-rhamnose. The nucleotides and phosphate residues contacted by RhaR were identified by chemical interference assays. All of the contacts are made to one face of the DNA and the symmetrical pattern matches the inverted repeat sequence proposed for the binding site. An unusual property of the binding site is that the two half-sites of the inverted repeat are separated from one another by 17 base-pairs of uncontacted DNA. Significant binding is retained if the 17 base-pairs are extended by insertions of integral turns of DNA, but not by half-integral turns. The complex of RhaR-DNA appears to be sharply bent, approximately 160 degrees.
Tobin
J F
JF
Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Schleif
R F
RF
D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005809:Genes, Regulator; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D012210:Rhamnose / Q000378:metabolism*
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; C062863:RhaR protein, E coli; D012210:Rhamnose