Origin of the asymmetrical contact between lac repressor and lac operator DNA.
The Escherichia coli lac operator DNA contains two sequence repeats related by a pseudo-dyad axis. Deviations from symmetry, in the central 21 bp sequence, occur at two pairs of symmetrically related sites (+15/+7, +13/+9) and at the central base-pair +11. Mutational analysis and DNA protection studies have suggested asymmetric interactions of lac repressor along this sequence. Previous biophysical studies on the lac repressor-operator system have typically employed symmetrized operator sequences to simplify analysis. As a result, it has remained difficult to assess the importance of the naturally occurring sequence deviations from symmetry. Here, 19F-NMR is used to determine if the wild-type E. coli lac operator DNA sequence itself specifies a pair of distinct half-site interactions with lac repressor DNA binding domains. To observe protein interactions simultaneously at operator half-sites using 19F-NMR, three pairs of naturally occurring, symmetry related thymine residues (at +6/+16, +8/+14 and +1/+21) were substituted pair-wise by 5-fluorodeoxyuridines (5-FdU). Two polypeptides corresponding to the N-terminal DNA binding domain of lac repressor "headpiece", residues 1 to 56 and 1 to 64, were employed to remove the steric constraints of subunit interaction in the wild-type tetramer. Spectral changes associated with headpiece binding to left side DNA sequences differ from those caused by binding to equivalent sequences on the right half-site. These results are similar to non-symmetric intact tetramer repressor interactions specified by the DNA sequence. Three mutant lac operator sequences with increased symmetry, bearing FdU substitutions were used to identify the relative importance of the three naturally asymmetric positions. Symmetrizing one pair of these sites alone or in addition to removing the central base-pair failed to produce identical NMR signal changes characteristic of symmetric headpiece-DNA complexes. However, symmetrizing both asymmetric pairs gave chemical shift changes expected from symmetric protein-DNA complexes. We propose that key interactions with the left side +9 (G.C) are altered at the symmetrically related right side +13 (A.T). The data show that the DNA sequence at +13 influences interactions three base-pairs away.
Rastinejad
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F
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Artz
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P
Lu
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P
D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism*; D003857:Deoxyuridine / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics*; D005461:Fluorine; D007554:Isotopes; D007763:Lac Operon / Q000235:genetics*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics*; D011487:Protein Conformation; D012097:Repressor Proteins / Q000378:metabolism*
D004269:DNA, Bacterial; D007554:Isotopes; D046911:Macromolecular Substances; D012097:Repressor Proteins; D005461:Fluorine; C061519:5-formyl-2'-deoxyuridine; D003857:Deoxyuridine