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Fermentable and nonfermentable carbon sources sustain constitutive levels of expression of yeast triosephosphate dehydrogenase 3 gene from distinct promoter elements.

1994; Elsevier BV; Volume: 269; Issue: 8 Linguagem: Inglês

10.1016/s0021-9258(17)37582-8

1083-351X

Shinya Kuroda, Sachiko Otaka, Yukio Fujisawa,

Enzyme Structure and Function

The triosephosphate dehydrogenase 3 gene (TDH3) is a glycolytic enzyme gene and is abundantly transcribed in Saccharomyces cerevisiae. The promoter region of the TDH3 gene is known to exhibit high transcriptional activity regardless of the fermentability of the carbon source and has been widely utilized to synthesize heterologous gene products in S. cerevisiae. To clarify the mechanism of constitutive transcription by the promoter, we constructed mutant promoters and analyzed the in vivo transcriptional activity of these promoters. The majority of the transcriptional potential is contained within a DNA fragment extending from nucleotides -524 to -255 (-524/-255; relative to the translation initiation codon), which consists of three cis-acting elements: a fermentable carbon source-dependent upstream activation sequence (UAS) 1 (-524/-426), a fermentable carbon source-dependent upstream repression sequence (URS) (-426/-393), and a nonfermetable carbon source-dependent UAS2 (-305/-255). This result indicates that the promoter involves two apparent promoter elements. One is fermentable carbon source-dependent, and another is nonfermentable carbon source-dependent. Southwestern analyses indicated that a novel 20-kDa protein is induced in yeast cells by shifting from a fermentable to nonfermentable carbon source. The protein interacts with two UAS1 13-base pair elements and one URS 13-base pair element, one of which had been previously designated GPE (general regulatory factor 1 (GRF1) binding site potentiator element) (Bitter, G. A., Chang, K. K. H., and Egan, K. M. (1991) Mol. Gen. Genet. 231, 22-32). We therefore termed the 20-kDa protein GPEB (GRF1-binding site potentiator element-binding protein). In addition, mutational analyses strongly suggested that UAS1, URS, and UAS2 interact with GRF1 and GPEB, GPEB, and the GCR1 (glycolysis regulation 1) gene product, respectively. We therefore concluded that constitutive transcription by the TDH3 promoter is sustained by two promoter elements and that the switch between them might be controlled by the nonfermentable carbon source-inducible GPEB.