Abstract
Structural variation of DNA within the promoter of the human Na, K-ATPase alpha2 gene, which contains a 35-base pair (bp) homopyrimidine.homopurine (Py.Pu) tract adjacent to a TATA box has been studied. The Py.Pu tract contains a 26-bp quasi-mirror repeat sequence with a potential for intramolecular triplex formation. As analyzed by two-dimensional agarose gel electrophoresis, a plasmid containing 151 bp of the promoter sequence including the 35-bp Py.Pu tract undergoes structural transitions under moderately acidic pH. Chemical probing with chloroacetaldehyde, dimethyl sulfate, and potassium permanganate is consistent with the formation of triplex DNA within the Py.Pu tract at native superhelical density as isolated from Escherichia coli. Chemical probing was used to determine a supercoil dependence for the formation of this combined unwound structure. At the superhelical density sufficient to locally unwind DNA, an H-y3 isomer of intermolecular triplex likely forms. However, at higher superhelical tension an H-y5 structure forms in the Py.Pu tract, and with increasing supercoiling the local DNA unwinding extends into the abutting TATA box. The H-y5/open TATA box combination structure might be favorable at higher superhelical densities since it relaxes more supercoils. The possible involvement of the H-y5/open TATA box structure in transcription is discussed.
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