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  • Title: DNA sequence dependent and independent conformational changes in multipartite operator recognition by lambda-repressor.
    Author: Deb S, Bandyopadhyay S, Roy S.
    Journal: Biochemistry; 2000 Mar 28; 39(12):3377-83. PubMed ID: 10727231.
    Abstract:
    Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage lambda have several pairs of repressor binding sites (O(R)1-O(R)2, O(R)2-O(R)3, O(L)1-O(L)2, and O(L)2-O(L)3) separated by a variable number of base pairs, and thus, bacteriophage lambda is a model system for studying multipartite operator recognition by DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in O(R)1-O(R)2 and O(L)2-O(L)3 but much less so in O(R)2-O(R)3. Upon titration of lambda-repressor with single-operator sites O(R)1, O(R)2, and O(R)3, it was observed that the tryptophan fluorescence quenches to different degrees, suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamide quenching of tryptophan fluorescence of lambda-repressor bound to these single operators also shows different Stern-Volmer constants, supporting the above conclusions. Titration of lambda-repressor with oligonucleotides containing pairs of operator sites also causes different degrees of fluorescence quenching. In particular, fluorescence quenching induced by O(R)1-O(R)2 binding is less than the quenching induced by either of the single operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescence of lambda-repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, O(R)2-O(R)3-bound repressor has significantly higher acrylamide quenchable components than either of the O(R)2- or O(R)3-bound proteins, again suggesting additional conformational changes upon establishment of protein-protein contact. We conclude that the strategy of recognition of multipartite operator by lambda-repressor is complex and varied, involving conformational changes in both DNA and protein that are determined by the separation of the binding sites as well as the nucleic acid sequence.
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