The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (73182 to 73133) and proximal (7177 to 781) p53-binding elements within which residues 73182 to 73151 (distal) and 7176 to 7149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we ®nd that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was con®rmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-de®-cient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.

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