Localization of a transcription promoter within the second exon of the cytochrome P-450c27/25 gene for the expression of the major species of two-kilobase mRNA

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Abstract
The rat P-450c27/25 (CYP27) gene is expressed as two distinctly sized mRNAs of 2 and 2.3 kb (kilobase). The 2 kb mRNA is the predominant form in the liver with negligible 2.3 kb species. Rat kidney and hepatoma, on the other hand, contain significant levels of the 2.3 kb species. Rat CYP27 gene contains 11 exons of 80-415 nucleotides that are separated by 10 introns of 83 bases to approximately 10 kb. S1 nuclease protection and primer extension analyses using liver RNA showed a prominent 5' terminus 86 nucleotides downstream from the start of exon 2. This site, designated as +1, is the start site for the 2 kb mRNA. 5' RACE analysis of rat kidney and hepatoma RNAs showed the presence of a 5' extended mRNA with a sequence complementary to the Spi2 mRNA. A cryptic TATA box (TTTAAA) is located 24 nucleotides upstream of the 2 kb mRNA transcription initiation site at +1. A 106 bp DNA fragment (sequence -83 to +23) that houses the putative TATA motif forms three differently migrating complexes with nuclear extract from the murine 3T3 cells. DNAse I footprinting and competition with synthetic DNA showed that complex A represents the bound Sp1 factor and complexes B and C are due to unknown factors binding to the -83 to -71 and -20 to -12 sequences, respectively. In vivo transcription analysis using -840/+23 DNA and its 5' deletions cloned in a CAT reporter plasmid suggests that the basal promoter elements are located within sequence -45 to +23 of the gene. Finally, in vitro transcription analysis in HeLa cell nuclear extract showed that intact TTTAAA motif and complex C-forming sequence from this region are essential for transcription initiation at the +1 position of the promoter. Our results demonstrate that the 2 kb mRNA is transcribed as an independent transcript driven by an immediate upstream promoter located within exon 2.

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