DEFINITION OF A CONSENSUS DNA-BINDING SITE FOR THE ESCHERICHIA-COLI PLEIOTROPIC REGULATORY PROTEIN, FRUR

The FruR regulator of Escherichia coli controls the initiation of tran scription of several operons encoding a variety of proteins involved i n carbon and energy metabolism. The sequence determinants of the FruR- binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding s ites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method ( BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was de monstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'GNNGAAT C/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranu cleotide is a typical motif of the DNA-binding site of the regulators of the GalR-LacI family. On the other hand, the high degree of degener acy found in the right half-site of this palindrome-like structure ind icated that FruR, which is a tetramer in solution, interacts asymmetri cally with the two half-sites of its operator. However, potentially Fr uR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA g ene, encoding phosphofructokinase-1, which is negatively regulated by FruR.