Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase kinase [corrected].
The rat branched-chain-2-oxo-acid dehydrogenase (BCOD) kinase mRNA is transcribed from a TATA-less promoter that has GC-rich sequences and two putative Sp1 binding sites near the transcription start site. We demonstrated previously that the 5' region of the kinase gene, base pairs -128 to +264, contained promoter activity when assayed using luciferase as a reporter (Huang and Chuang (1996) Biochem. J. 313, 603-609). To define DNA elements required for efficient expression of the kinase gene, nested deletion constructs of the above promoter region fused with a luciferase reporter gene were transfected into cultured H4IIE (hepatoma) and NRK-52E (kidney) cells. The results showed that the region between nucleotides -58 and +21 was indispensable for the kinase basal promoter activity. Methylation-interference and mutagenesis-promoter assays identified nucleotides -50 to -40 (ACAACTCCCA) as cis-acting DNA sequences that are required for nuclear protein binding and efficient promoter activity. Gel-supershift analysis with anti-Sp1 antibody suggested that the nuclear protein capable of binding to the -58 oligonucleotide (bp -58 to -34) was immunologically related to the Sp1 protein. The -58 oligonucleotide formed a DNA-protein complex with recombinant Sp1 protein with an affinity approximately ten-fold lower than that of the consensus Sp1 oligonucleotide. Co-transfection of the Sp1 expression plasmid and the -58 promoter construct into Drosophila Schneider cells revealed that Sp1 contributed to the kinase basal promoter activity by binding to the non-consensus site in the -58 region. Deletion of two consensus Sp1 binding sites (bases -150 to -140 and bases +29 to +38) in the kinase gene did not affect the basal promoter activity. Therefore binding of Sp1 or Sp1-like proteins to the above single non-consensus Sp1 sequence in the -58 region plays a major role of transactivating basal expression of the BCOD kinase.
Huang
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YS
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9038, USA.
Chuang
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DT
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004330:Drosophila; D015870:Gene Expression; D017930:Genes, Reporter; D007658:Ketone Oxidoreductases / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000235:genetics*; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D017931:DNA Primers; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D004247:DNA; D008156:Luciferases; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)