Generation and statistical mechanical modeling of Z-DNA in the mouse metallothionein I promoter.
The mouse metallothionein I promoter has a potential Z-DNA sequence 86-102 bases upstream from the transcription start. The alternating purine/pyrimidine nature of this sequence, d-(GCGCGTGACTATGCGTG), is interrupted by a central d(AC) dinucleotide. We reversed the d(AC) to d(CA) by site-directed mutagenesis and subcloned the wild-type and mutated 218 base pair (bp) SstI-BglII fragment containing this region into pUC12. By two-dimensional chloroquine gel analysis, the mutant promoter clearly underwent a B-Z transition with a resulting loss of at least five twists. Purines within this sequence exhibited diethyl pyrocarbonate (DEP) sensitivity, which extended in both the 5' and 3' direction encompassing a region of approximately 32 base pairs. When subjected to sufficient torsional stress, the wild-type sequence showed weak evidence of a transition on chloroquine gels and clear DEP sensitivity with a similar, yet distinct pattern. Statistical mechanical modeling of the chloroquine gel analysis demonstrated that the average free energy of propagation for the mutant sequence (0.7-0.9 kcal mol-1 bp-1) was approximately twice that for d(CG)n sequences and that the sequence d(GACGCGGGGCGCGTGCATATGCGTGG) forms the core Z-DNA region.
Visentin
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Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Harley
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CB
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D005796:Genes; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013816:Thermodynamics
D004247:DNA; D008668:Metallothionein