The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene (MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, electrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations -51/-33, -267/-249 and -555/-526. The most distal and proximal domains are positive-acting, whereas the element at -267/-249 acts in a negative manner. The positive-acting -51/-33 element contains the middle of three consensus Sp1-recognition sequences found in this region of the gene. Binding of Y1 cell nuclear protein to a DNA fragment corresponding to this region of the gene is competed by a synthetic oligodeoxyribonucleotide bearing the consensus Sp1-binding site sequence. These studies provide the first reported functional evidence localizing transcriptional elements of MBR.
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