Structural organization of the rat branched-chain 2-oxo-acid dehydrogenase kinase gene and partial characterization of the promoter-regulatory region

The gene encoding the rat branched-chain 2-oxo-acid dehydrogenase kinase (EC 2.7.1.115) has been isolated and partially characterized. The entire gene, including the promoter-regulatory region, spans 6 kb and contains 11 exons. The 5'-untranslated region comprising 264 bp is interrupted by intron 1 which is 581 bp in size. The complete in-frame sequence of intron 7 encodes the 49 amino acid insert previously reported to be present in the larger isoform of the rat kinase. Sequencing of the 679 bp of the 5'-flanking region showed the absence of a canonical TATA box, similar to other branched-chain 2-oxo-acid dehydrogenase-complex genes. Several candidate cis-acting elements are present. These include CAAT boxes, Sp-1-binding sites, GCN-4 sites, CCAAT enhancer binding-protein sites (C/EBP) and glucocorticoid-responsive element (GRE) sites. Also present are a pair of direct repeats of unknown function. The luciferase-reporter assay showed that promoter activity is markedly higher in normal rat kidney (NRK-52E) cells than in rat hepatoma (FTO-2B) cells, and that the 5'-flanking region between bases -449 and +264 is both necessary and sufficient for basal transcription of the kinase gene.