Abstract
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.
Highlights
Escherichia coli Lac repressor (LacR) is a transcriptional regulatory protein; LacR functions by binding, in the absence of the inducer molecules allolactose or isopropyl-/3-Dthiogalactoside, to a specific DNA site, lac operator, located adjacent to the lac promoter
LacR is a tetramer of four chemically identical subunits, each consisting of 360 amino acids
The DNA binding domain of LacR can be cleaved proteolytically from the remainder of the molecule to yield a 56-residue fragment that folds independently and that binds to the specific DNA site [6,7,8]
Summary
Escherichia coli Lac repressor (LacR) is a transcriptional regulatory protein; LacR functions by binding, in the absence of the inducer molecules allolactose or isopropyl-/3-Dthiogalactoside, to a specific DNA site, lac operator, located adjacent to the lac promoter (reviewed in ref. 1). The 56 N-terminal amino acids constitute the DNA binding domain of LacR. The left DNA half site for LacR is 5'-AATTGTGAGC-3'.
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