DNA Structure Determines Protein Binding and Transcriptional Efficiency of the Proenkephalin cAMP-responsive Enhancer

Cynthia T. McMurray,Craig Spiro,Xiling Wu,David P. Bazett-Jones

doi:10.1074/jbc.270.46.27702

Cynthia T. McMurray, Craig Spiro + Show 2 more

Open Access

https://doi.org/10.1074/jbc.270.46.27702

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Abstract

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.

Highlights

A limited number of sequence-specific DNA-binding proteins is sufficient for the precise and complex transcriptional regulation in the brain
What mechanism requires that the arrangement of cAMP response elements (CREs)-1 and CRE-2 be precisely maintained? A direct role for CRE-1 in the action of CRE-binding proteins is suggested by the observation that response to CREB protein is altered when CRE-2 is intact but CRE-1 is deleted [9]
About 90% of proenkephalin molecules were free of bound protein, whereas approximately 10% contained a single complex in a position consistent with binding at the CREs (Fig. 1, b and e)

Summary

Introduction

A limited number of sequence-specific DNA-binding proteins is sufficient for the precise and complex transcriptional regulation in the brain. Binding site differs from that of CRE-2 by the presence of two GT mismatched base pairs [9, 18, 19], and it binds CREB protein with higher affinity than does the native duplex site [9]. To test the biological relevance of the alternative site, we have studied protein-proenkephalin complexes in very large molecules of DNA. In the alternative binding site, both CRE-1 and CRE-2 would directly interact with CREB protein Both CREs should bind protein during the formation of active transcription complexes. An alternative conformation of the DNA creates a single protein binding site required for the proenkephalin-specific transcriptional machine

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