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A CCAAT DNA binding factor consisting of two different components that are both required for DNA binding
A Hatamochi1, P T Golumbek, E Van Schaftingen
1Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
The Journal of Biological Chemistry
|April 25, 1988
Summary
Researchers purified a novel CCAAT-binding complex from rat liver and fibroblast nuclear extracts. This complex, comprising heat-sensitive factor A and heat-resistant factor B, is essential for DNA binding to the alpha 2(I) collagen promoter.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- CCAAT-binding proteins play crucial roles in gene regulation.
- Understanding these factors is key to deciphering gene expression control.
Purpose of the Study:
- To purify and characterize a novel CCAAT-binding activity from mammalian nuclear extracts.
- To determine the molecular properties and DNA-binding requirements of this activity.
Main Methods:
- Purification of nuclear factors using DNA-binding assays with the mouse alpha 2(I) collagen promoter.
- Ion exchange chromatography (Mono Q/S), heparin-agarose, and affinity chromatography.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by renaturation to determine molecular weights.
Main Results:
- A CCAAT-binding activity was resolved into two components, heat-sensitive factor A (Mr 39,000) and heat-resistant factor B (Mr 41,000).
- Both factors A and B are necessary for DNA binding and form a complex (A + B).
- Competition experiments revealed a unique DNA sequence specificity distinct from known CCAAT-binding proteins.
Conclusions:
- A novel CCAAT-binding complex composed of two distinct protein factors has been purified.
- This complex exhibits unique DNA sequence recognition, suggesting a specific role in gene regulation.
- Further characterization of this complex will elucidate its function in controlling gene expression.