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 expression of many operons in Escherichia coli including several involved in the metabolism of the branched-chain amino acids, L-isoleucine, L-valine, and L-leucine. The ilvGMEDA operon contains the genes for four of the five enzymes of the common pathway for the biosynthesis of these amino acids. A high affinity, consensus-like Lrp-DNA binding site has been identified at an unusual position in the leader region of this operon 226 base pairs downstream of the transcriptional initiation site between the attenuator and the ilvG gene. Binding to this site facilitates the cooperative binding of a second Lrp protomer to an adjacent, upstream, secondary site. At higher Lrp concentrations, binding to a third site is observed. Chemical, enzymatic, and alkylation protection and interference footprinting experiments demonstrate that the Lrp homodimer contacts the DNA helix at symmetrical half-sites present in adjacent 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-attenuator region of the ilvGMEDA operon. Lrp-dependent production of attenuated 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 the regulation of ilvGMEDA operon expression is discussed.
Rhee
K Y
KY
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA.
Parekh
B S
BS
Hatfield
G W
GW
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D051756:Leucine-Responsive Regulatory Protein; D009876:Operon; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D021382:Protein Sorting Signals; D014157:Transcription Factors; D051756:Leucine-Responsive Regulatory Protein