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 c AMP response elements (CRE-1 and CRE-2) are needed to mediate the tran scriptional response to cAMP and to the CRE-binding protein (CREB) tra nscription factor. CRE-1 and CRE-2 are contained within a nearly palin dromic region that can form stable hairpin structures in vitro. CREB b inds 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 h airpin 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 f or high-affinity binding. The existence within a short enhancer of alt ernative sites for a single factor suggests a mechanism for regulation of transcription by DNA structure.