LEUCINE-RESPONSIVE REGULATORY PROTEIN-DNA INTERACTIONS IN THE LEADER REGION OF THE ILVGMEDA OPERON OF ESCHERICHIA-COLI

The leucine-responsive regulatory protein (Lrp) regulates the expressi on of many operons in Escherichia coil including several involved in t he metabolism of the branched-chain amino acids, L-isoleucine, L-valin e, and L-leucine. The ilvGMEDA operon contains the genes for four of t he five enzymes of the common pathway for the biosynthesis of these am ino acids, A high affinity, consensus-like Lrp-DNA binding site has be en identified at an unusual position in the leader region of this oper on 226 base pairs downstream of the transcriptional initiation site be tween the attenuator and the ilvG gene, Binding to this site facilitat es the cooperative binding of a second Lrp protomer to an adjacent, up stream, secondary site, At higher Lrp concentrations, binding to a thi rd site is observed, Chemical, enzymatic, and alkylation protection an d interference footprinting experiments demonstrate that the Lrp homod imer contacts the DNA helix at symmetrical half-sites present in adjac ent major grooves and that the primary and secondary binding sites are separated by one helical turn and aligned along the same face of the DNA helix, In vivo, Lrp represses transcription through the leader-att enuator region of the ilvGMEDA operon, Lrp-dependent production of att enuated RNA transcripts is also observed in vitro. No transcriptional effects are observed, in vivo or in vitro, in the absence of an intact Lrp primary binding site, A possible physiological role for Lrp in th e regulation of ilvGMEDA operon expression is discussed.