Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase

Yi Shuian Huang, David T. Chuang

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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. 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.

Original languageEnglish (US)
Pages (from-to)713-722
Number of pages10
JournalBiochemical Journal
Volume334
Issue number3
StatePublished - Sep 15 1998

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Rats
Phosphotransferases
Genes
Oligonucleotides
Nuclear Proteins
Luciferases
Protein Binding
Nucleotides
Binding Sites
GC Rich Sequence
Recombinant proteins
Mutagenesis
Proteins
Methylation
Transcription Initiation Site
DNA sequences
DNA
Gene Deletion
Reporter Genes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism for basal expression of rat mitochondrial branched-chain-2-oxo-acid dehydrogenase. / Huang, Yi Shuian; Chuang, David T.

In: Biochemical Journal, Vol. 334, No. 3, 15.09.1998, p. 713-722.

Research output: Contribution to journalArticle

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