Hairpin formation within the human enkephalin enhancer region. 1. Kinetic analysis.
The 3'-5' cyclic AMP inducible enhancer region of the human enkephalin gene is located within an imperfect palindrome of 23 base pairs, located from -106 to -84 base pairs upstream of the transcriptional start site. Recent evidence has indicated that hairpin formation within this region may be involved in transcriptional regulation of the human proenkephalin gene. A 23-bp synthetic oligonucleotide of this region has been shown to undergo a reversible conformational change from a duplex to a cruciform structure of two hairpins [McMurray, C.T., Wilson, W.D., & Douglass, J.O. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 666]. Our current studies explore the kinetics and activation energies of the hairpin to duplex transitions of synthetic oligonucleotides under a variety of conditions. Ultraviolet spectroscopic data collected over a range of pH values, ionic strengths, and temperatures are used to determine the reaction rates and activation energies of the hairpin to duplex reaction. The rate of formation of a duplex from two hairpins is a slow second-order process, dependent on both pH and ionic strength. The return from the hairpin state to the duplex state occurs with a high activation energy of 22-41 kcal/mol strand, depending on the conditions. Pseudo-first-order reaction conditions indicate that one of the hairpins, the AC hairpin, is the rate-limiting reactant. These results suggest a model by which the formation of a cruciform might regulate transcription.
Gacy
A M
AM
Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905.
McMurray
C T
CT
D001483:Base Sequence; D001665:Binding Sites; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000737:chemistry*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004745:Enkephalins / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D011498:Protein Precursors / Q000235:genetics*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D004247:DNA