Secondary structure creates mismatched base pairs required for high-affinity binding of cAMP response element-binding protein to the human enkephalin enhancer.
Transactivation studies of the enkephalin enhancer indicate that two cAMP response elements (CRE-1 and CRE-2) are needed to mediate the transcriptional response to cAMP and to the CRE-binding protein (CREB) transcription factor. CRE-1 and CRE-2 are contained within a nearly palindromic region that can form stable hairpin structures in vitro. CREB binds only weakly to the native duplex enhancer and only within CRE-2. In contrast, CREB binds with high affinity to the hairpin in which CRE-1 and CRE-2 come together to form a CREB site with two G.T base pairs. NMR and binding studies show that high-affinity binding to the G.T hairpin requires one of the mismatched G.T pairs. Insertion of that G.T pair into the duplex confers high-affinity binding. Parallel studies with the somatostatin CRE show that the T in one G.T pair is crucial for high-affinity binding. The existence within a short enhancer of alternative sites for a single factor suggests a mechanism for regulation of transcription by DNA structure.
Department of Pharmacology, Mayo Foundation, Rochester, MN 55905.
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C
Spiro
J P
JP
Richards
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S
Chandrasekaran
R G
RG
Brennan
C T
CT
McMurray
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D004745:Enkephalins / Q000235:genetics*; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004745:Enkephalins; D009838:Oligodeoxyribonucleotides; D011953:Receptors, Cyclic AMP