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An analysis, performed by DNase I footprinting, of the interactions between factors present in Molt-4 nuclear extracts and a Xenopus U2 snRNA gene promoter is presented. Four distinct regions of sequence-specific DNA-factor interaction are found. Two of these correspond to the previously identified proximal and distal sequence elements (PSE and DSE) of the promoter. Both of these elements are important in U2 transcription, indicating a functional role for the observed interactions. The other two sites of interaction correspond to a sequence element conserved in many, but not all, vertebrate U snRNA gene promoters (the MSE) and to a region adjacent to the site of transcription initiation (the "cap site"). Site-directed mutants of these latter two elements are constructed which no longer bind nuclear factors. Transcriptional analysis in Xenopus oocytes reveals that these mutants are transcribed as efficiently as wild-type U2. Other possible roles for the two factors are discussed.