Orientation of the Lac repressor DNA binding domain in complex with the left lac operator half site characterized by affinity cleaving.
Lac repressor (LacR) is a helix-turn-helix motif sequence-specific DNA binding protein. Based on proton NMR spectroscopic investigations, Kaptein and co-workers have proposed that the helix-turn-helix motif of LacR binds to DNA in an orientation opposite to that of the helix-turn-helix motifs of lambda repressor, lambda cro, 434 repressor, 434 cro, and CAP [Boelens, R., Scheek, R., van Boom, J. and Kaptein, R., J. Mol. Biol. 193, 1987, 213-216]. In the present work, we have determined the orientation of the helix-turn-helix motif of LacR in the LacR-DNA complex by the affinity cleaving method. The DNA cleaving moiety EDTA.Fe was attached to the N-terminus of a 56-residue synthetic protein corresponding to the DNA binding domain of LacR. We have formed the complex between the modified protein and the left DNA half site for LacR. The locations of the resulting DNA cleavage positions relative to the left DNA half site provide strong support for the proposal of Kaptein and co-workers.
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
J A
JA
Shin
R H
RH
Ebright
P B
PB
Dervan
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D004269:DNA, Bacterial / Q000378:metabolism*; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007763:Lac Operon / Q000502:physiology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D012097:Repressor Proteins; C071212:lac repressor (1-56), ((EDTA-Fe)-gamma-aminobutyryl)-; D004492:Edetic Acid