Interaction of the trp repressor with trp operator DNA fragments.
The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor L-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpOc, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the 31P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.
Beckmann
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P
Laboratory of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London.
Martin
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SR
Lane
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AN
D001483:Base Sequence; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics / Q000502:physiology*; D010758:Phosphorus; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D010758:Phosphorus; D014364:Tryptophan