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.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Exons/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Animals , Base Sequence , Blotting, Northern , Carcinoma, Hepatocellular , Cell Line , Cytochrome P-450 Enzyme System/chemistry , DNA Primers , DNA-Binding Proteins/metabolism , Deoxyribonuclease I/metabolism , Genes, Reporter/genetics , Kidney , Molecular Sequence Data , Rats , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Single-Strand Specific DNA and RNA Endonucleases/metabolism , TATA Box/genetics , Transcription, Genetic/genetics , Tumor Cells, CulturedABSTRACT
The murine cytochrome c oxidase (COX) subunit Vb mRNA contains heterogeneous 5' ends mapping to +1, +6, +12, +17-22, +24-29, and +32-35 positions of the gene. We have previously shown that initiation of RNA at the +1 position of the promoter depends upon a YY-1 (NF-E1) binding initiator motif. In this article we show that the GABP factor binding duplicated ets motif, GTTCCCGGAAG, at +16 to +26 position functions as an independent initiator for transcription of RNAs mapping to the +18-19 and +23-26 regions. The initiation region ets sequence repeat (ets-ets sequence) can drive the transcription of the CAT reporter gene. The upstream ets site of the ets-ets sequence exhibits a low affinity for binding to purified GABP factors, whereas the downstream site exhibits high affinity. S1 analyses of RNA from transfected COS cells demonstrate that the initiation region ets-ets sequence can accurately initiate transcription at the +18-19 and +24-25 regions. Transcriptional initiation at these two positions, but not at +1, +12, and +31-32 positions, show a selective dependence for intact downstream ets site and GABP alpha and beta factors as tested in an in vitro reconstituted system. The activities of both COX IV and COX Vb single site ets initiators are induced in vivo by coexpression with GABP alpha and beta cDNAs. These results provide evidence that the 5' heterogeneity of the COX Vb mRNA is largely due to independent transcription initiators at multiple initiator motifs that bind to various transcription factors.