DNA Sequence Dependent and Independent Conformational Changes in Multipartite Operator Recognition by -Repressor
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- 作者：Sunanda Deb ; Sumita Bandyopadhyay ; and Siddhartha Roy
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：March 28, 2000
- 年：2000
- 卷：39
- 期：12
- 页码：3377 - 3383
- 全文大小：70K
- 年卷期：v.39,no.12(March 28, 2000)
- ISSN：1520-4995
文摘
Binding of regulatory proteins to multipartite DNA binding sites often occurs with protein-protein interaction, resulting in cooperative binding. The operators of bacteriophage 
have several pairsof repressor binding sites (OR1-OR2, OR2-OR3, OL1-OL2, and OL2-OL3) separated by a variable numberof base pairs, and thus, bacteriophage is a model system for studying multipartite operator recognitionby DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in OR1-OR2 and OL2-OL3 but much less so in OR2-OR3. Upon titration of -repressor with single-operator sitesOR1, OR2, and OR3, it was observed that the tryptophan fluorescence quenches to different degrees,suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamidequenching of tryptophan fluorescence of -repressor bound to these single operators also shows differentStern-Volmer constants, supporting the above conclusions. Titration of -repressor with oligonucleotidescontaining pairs of operator sites also causes different degrees of fluorescence quenching. In particular,fluorescence quenching induced by OR1-OR2 binding is less than the quenching induced by either of thesingle operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescenceof -repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, OR2-OR3-bound repressor has significantly higher acrylamidequenchable components than either of the OR2- or OR3-bound proteins, again suggesting additionalconformational changes upon establishment of protein-protein contact. We conclude that the strategy ofrecognition of multipartite operator by -repressor is complex and varied, involving conformational changesin both DNA and protein that are determined by the separation of the binding sites as well as the nucleicacid sequence.
have several pairsof repressor binding sites (OR1-OR2, OR2-OR3, OL1-OL2, and OL2-OL3) separated by a variable numberof base pairs, and thus, bacteriophage is a model system for studying multipartite operator recognitionby DNA-binding proteins. Near-UV circular dichroism spectra show that the DNA is distorted in OR1-OR2 and OL2-OL3 but much less so in OR2-OR3. Upon titration of -repressor with single-operator sitesOR1, OR2, and OR3, it was observed that the tryptophan fluorescence quenches to different degrees,suggesting different conformations of the protein in the three DNA-protein complexes. Acrylamidequenching of tryptophan fluorescence of -repressor bound to these single operators also shows differentStern-Volmer constants, supporting the above conclusions. Titration of -repressor with oligonucleotidescontaining pairs of operator sites also causes different degrees of fluorescence quenching. In particular,fluorescence quenching induced by OR1-OR2 binding is less than the quenching induced by either of thesingle operators alone, suggesting additional conformational changes upon establishment of protein-protein contact. Stern-Volmer constants obtained from acrylamide quenching of tryptophan fluorescenceof -repressor bound cooperatively to pairs of operator sites are different from those of the single-operator-site-bound repressors. For example, OR2-OR3-bound repressor has significantly higher acrylamidequenchable components than either of the OR2- or OR3-bound proteins, again suggesting additionalconformational changes upon establishment of protein-protein contact. We conclude that the strategy ofrecognition of multipartite operator by -repressor is complex and varied, involving conformational changesin both DNA and protein that are determined by the separation of the binding sites as well as the nucleicacid sequence.