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 Zero-acid dehydrogenase kinas e (EC 2.7.1.115) has been isolated and partially characterized. The en tire gene, including the promoter-regulatory region, spans 6 kb and co ntains 11 exons. The 5'-untranslated region comprising 264 bp is inter rupted by intron I which is 581 bp in size. The complete in-frame sequ ence of intron 7 encodes the 49 amino acid insert previously reported to be present in the larger isoform of the rat kinase (Harris, Popov, Shimomura, Zhao, Jaskiewicz, Nanaumi and Suzuki (1992) Adv. Enzyme Reg ul. 32, 267-284). Sequencing of the 679 bp of the 5'-flanking region s howed the absence of a canonical TATA box, similar to other branched-c hain 2-oxo-acid dehydrogenase-complex genes. Several candidate cis-act ing elements are present. These include CAAT boxes, Sp-1-binding sites , GCN-4 sites, CCAAT enhancer binding-protein sites (C/EBP) and glucoc orticoid-responsive element (GRE) sites. Also present are a pair of di rect repeats of unknown function. The luciferase-reporter assay showed that promoter activity is markedly higher in normal rat kidney (NRK-5 2E) cells than in rat hepatoma (FTO-2B) cells, and that the 5'-flankin g region between bases -449 and +264 is both necessary and sufficient for basal transcription of the kinase gene.