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Related Experiment Videos

Binding constant determination studies utilizing recombinant delta CREB protein

J S Williams1, J E Dixon, O M Andrisani

  • 1Department of Veterinary Physiology and Pharmacology, Purdue University, W. Lafayette, IN 47907-1246.

DNA and Cell Biology
|March 1, 1993
PubMed
Summary

This study quantifies the binding affinity of the cAMP-responsive element binding protein (delta-CREB) for different DNA sequences. Delta-CREB binds strongly to natural cAMP-response element (CRE) motifs in gene promoters.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • The cAMP-responsive element binding protein (CREB) family regulates gene expression.
  • Understanding the binding specificities of CREB variants like delta-CREB is crucial for deciphering gene regulation.
  • Quantitative binding data for delta-CREB to various cAMP-response element (CRE) motifs are limited.

Purpose of the Study:

  • To determine the binding constants (Kd) of purified recombinant delta-CREB for natural and variant CRE DNA motifs.
  • To quantitatively assess the in vitro binding affinity of delta-CREB to specific CRE sequences found in neuropeptide hormone gene promoters.

Main Methods:

  • Purified recombinant delta-CREB protein was used in binding reactions.
  • Binding affinities were measured against natural CRE motifs from somatostatin and enkephalin gene promoters.

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  • Binding affinities were also tested against several variant CRE motifs.

    • Main Results:

    • The binding constant (Kd) of delta-CREB for the palindromic CRE (TGACG-TCA) in the somatostatin promoter was determined to be 5.0 x 10(-9) M.
    • The Kd for the variant CRE motif (TG_CGTCA) in the enkephalin promoter was calculated to be 3 x 10(-8) M.
    • These results provide quantitative in vitro binding data for delta-CREB.

    Conclusions:

    • Delta-CREB exhibits distinct binding affinities for different CRE DNA motifs.
    • The quantitative data provide insights into the sequence specificity of delta-CREB binding.
    • This study contributes to understanding the molecular mechanisms of gene regulation by delta-CREB.