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The rat brain creatine kinase gene possesses a structurally complex promoter with multiple potential regulatory elements. Two CCAAT sequences, a TATAAATA sequence and a TTAA sequence are found within the first one hundred base pairs. We present evidence that favors the allocation of the downstream TTAA sequence as the potential TATA box. We show that the CCAAT sequences and the upstream TATAAATA sequence are binding sites for potential regulatory factors and that sequences in this region are capable of regulating expression from the downstream TTAA sequence. We suggest that the protein that binds to the upstream TATAAATA sequence is not a classical TFIID factor but rather may serve to block the binding of TFIID and/or to promote transcription from the downstream start site. We have been able to define conditions in vitro under which binding to this upstream TATAAATA sequence does not occur. Under these conditions we are able to detect transcription from both potential TATA sequences, a situation which we have been unable to detect in vivo. Our experiments suggest the existence in HeLa and brain nuclei of a protein that recognizes the concensus TATAAATA sequence, that is distinct from TFIID, and that may function in part to deny access of TFIID to this potential promoter element.

We have previously reported that the rat brain creatine kinase (ckb) gene promoter contains an AT-rich sequence that is a binding site for a protein called TARP (TA-rich recognition protein). This AT-rich segment is a positively acting regulatory element for the ckb promoter. A similar AT-rich DNA segment is found at the 3' end of the 5' muscle-specific enhancer of the rat muscle creatine kinase (ckm) gene and has been shown to be necessary for full muscle-specific enhancer activity. In this report, we show that TARP binds not only to the ckb promoter but also to the AT-rich segment at the 3' end of the muscle-specific ckm enhancer. A second, weaker TARP-binding site was identified in the ckm enhancer and lies at the 5' end of the minimal enhancer segment. TARP was found in both muscle cells (C2 and L6 myotubes) and nonmuscle (HeLa) cells and appeared to be indistinguishable from both sources, as judged by gel retardation and footprinting assays. The TARP-binding sites in the ckm enhancer and the ckb promoter were found to be functionally interchangeable. We propose that TARP is active in both muscle and nonmuscle cells and that it is one of many potential activators that may interact with muscle-specific regulators to determine the myogenic phenotype.

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