DNA structure determines protein binding and transcriptional efficiency of the proenkephalin cAMP-responsive enhancer.
Two precisely arranged proenkephalin cAMP response elements (CREs) behave as a single protein binding site. The experiments described support a model in which a secondary structural change creates a new binding site, which is made up of sequences from both of the CREs. The CRE-binding protein (CREB) binds CRE-1, but binding there is entirely dependent on the presence of CRE-2. Electron spectroscopic images show that a CREB dimer occupies twice as much DNA in the proenkephalin gene as in the prodynorphin gene. The enhancer region is sensitive to P1 nuclease in a CREB concentration-dependent manner, and sensitivity is strand-specific, indicating protein-stabilized structural change. DNase I analysis shows that in the native proenkephalin gene, CREB binds both CRE-1 and CRE-2. In vivo, both CREs are occupied in the transcriptionally active proenkephalin gene, while neither is in the silent gene. Whereas CREB can bind CRE-2, mutation or elimination of either proenkephalin CRE alters response to second messengers and transcription factors. Thus, binding to CRE-2 alone is not sufficient. Specific and efficient transcription of the proenkephalin gene requires the presence of both CREs, precisely arranged to allow them to form a single protein binding site.
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D002846:Chromatography, Affinity; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004745:Enkephalins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D014158:Transcription, Genetic
D000906:Antibodies; D017362:Cyclic AMP Response Element-Binding Protein; D004745:Enkephalins; D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins; C029992:proenkephalin; D004247:DNA; C033660:preproenkephalin; D015500:Chloramphenicol O-Acetyltransferase