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Sequence-dependent differences in DNA structure influence the affinity of P22 operator for P22 repressor

L Wu1, G B Koudelka

  • 1Department of Biological Sciences, State University of New York, Buffalo 14260.

The Journal of Biological Chemistry
|September 5, 1993
PubMed
Summary

DNA sequence at the P22 operator

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetics

Background:

  • The P22 repressor protein regulates gene expression by binding to the P22 operator DNA sequence.
  • The precise mechanisms by which DNA sequence influences repressor-operator binding affinity are not fully understood.
  • Previous studies suggest that while direct contact with central bases is absent, sequence still impacts affinity.

Purpose of the Study:

  • To investigate how central base pair sequences in the P22 operator affect repressor binding affinity.
  • To elucidate the structural and dynamic basis for sequence-dependent affinity variations.
  • To determine the role of DNA torsional flexibility and deformability in protein-DNA interactions.

Main Methods:

  • Chemical probing using KMnO4 and hydroxyl radical (.OH) to assess DNA accessibility.
  • Ring closure studies to measure DNA torsional properties (twist).
  • Analysis of DNA minor groove width in the presence and absence of repressor protein.

Main Results:

  • Operator DNA with central C-G bases exhibits lower repressor affinity than operator DNA with central T-A bases.
  • The minor groove of the lower-affinity C-G operator is wider than that of the higher-affinity T-A operator, irrespective of repressor binding.
  • Central sequences influence operator DNA's inherent twist, with C-G operators being more overtwisted than T-A operators in solution.
  • P22 repressor binding unwinds both operator types to a similar extent, maintaining their relative twist differences.
  • DNA torsional flexibility does not dictate binding affinity; instead, central sequences limit operator deformability within the repressor-operator complex.

Conclusions:

  • Central DNA sequences modulate P22 repressor-operator affinity not through torsional flexibility but by restricting DNA deformability.
  • The stability of the repressor-operator complex is influenced by sequence-dependent alterations in protein-DNA contacts.
  • Operator sequence dictates minor groove width and inherent DNA twist, impacting how effectively the DNA can be deformed to fit the repressor binding site.

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