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Related Experiment Videos

A new DNA binding mode for CAP.

J M Hudson1, L G Crowe, M G Fried

  • 1Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.

The Journal of Biological Chemistry
|February 25, 1990
PubMed
Summary
This summary is machine-generated.

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Escherichia coli cyclic AMP receptor protein (CAP) binds DNA fragments without sequence specificity. This interaction shows less cooperativity than previously reported, suggesting unknown DNA elements influence CAP binding.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The cyclic AMP receptor protein (CAP) is a transcriptional regulator in Escherichia coli.
  • CAP requires cyclic AMP (cAMP) for DNA binding and transcriptional activation.
  • Previous studies indicated CAP exhibits high cooperativity when binding to genomic DNA.

Purpose of the Study:

  • To investigate the binding characteristics of CAP to specific DNA fragments in the absence of cAMP.
  • To determine the equilibrium constant and cooperativity parameter for CAP-DNA complex formation.
  • To explore the potential influence of DNA structure on CAP binding cooperativity.

Main Methods:

  • Electrophoresis mobility shift assay (EMSA) was employed to study CAP-DNA interactions.
  • Restriction fragments containing lac and crp promoter sequences were used.

Related Experiment Videos

  • Equilibrium binding constants and cooperativity parameters were estimated under standard buffer conditions.

    • Main Results:

    • CAP binding to restriction fragments was observed without detectable sequence specificity.
    • The estimated equilibrium constant was 114,000 +/- 1400 M-1, with a cooperativity parameter of 1.3 +/- 0.8.
    • CAP-DNA complexes showed uniform mobility decrements, suggesting minimal DNA bending.
    • Binding cooperativity differed significantly between restriction fragments and genomic DNA.

    Conclusions:

    • CAP binding to specific DNA fragments in vitro lacks high sequence specificity and cooperativity.
    • The reduced cooperativity compared to genomic DNA suggests modulation by other DNA sequences or structures.
    • These findings highlight the potential for DNA context to influence CAP's regulatory function.