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Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties.
J C Craig1, M A Schumacher, S E Mansoor
1Vollum Institute and Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098, USA.
The Journal of Biological Chemistry
|January 13, 2001
Summary
The cAMP response element-binding protein (CREB) uses specific interactions and magnesium ions for DNA binding. These mechanisms differ for consensus versus variant CRE sequences, suggesting distinct binding pathways.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- The cAMP response element-binding protein (CREB) is a transcription factor with conserved dimerization and DNA binding features.
- Its basic leucine zipper (bZIP) domain mediates these interactions, influenced by specific amino acid residues and ions.
Purpose of the Study:
- To investigate the roles of specific amino acid residues (Tyr307, Glu319, Lys304) and magnesium ions in CREB dimerization and DNA binding.
- To elucidate the mechanisms by which CREB interacts with both consensus and variant CRE sequences.
Main Methods:
- Site-directed mutagenesis of CREB to alter key amino acid residues (Y307F, Y307R, E319A, E319K, K304A).
- Assessing dimer stability and DNA binding affinity using biochemical assays.
- Evaluating the effect of magnesium ions on CREB binding to palindromic (consensus) and variant CRE sequences.
Main Results:
- Mutations at Tyr307 and Glu319 affected CREB dimer stability and DNA binding affinity.
- Magnesium ions significantly enhanced wild-type CREB binding to palindromic CREs but not variant CREs.
- The K304A mutation abolished CREB binding to palindromic CREs while retaining binding to variant CREs.
Conclusions:
- CREB dimerization and DNA binding are modulated by specific amino acid interactions and magnesium ions.
- Magnesium ions differentially regulate CREB binding to consensus and variant CRE sequences.
- CREB likely employs distinct mechanisms for binding to consensus and variant CRE sequences.