Abstract
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.
Highlights
Fermentable and Nonfermentable Carbon Sources Sustain Constitutive Levels of Expression of Yeast Triosephosphate Dehydrogenase3 Gene from Distinct Promoter Elements”
Source-dependent upstream activationsequence (UAS) For the HBsAg L protein and hepatitisB virus core particles, 1 (-5241-426), a fermentable carbon source-depend- the amount of synthesized protein represented more than ent upstream repressionsequence (URS) (-426/-393), 40% of the soluble yeast protein. and a nonfermentable carbonsource-dependent UAS2 It has been reported that the steady-state level of TDH3 (-3051-255)
We show that constitutive transcription by the TDH3 promoter is sustained by two promoter elementas,fermentable carbon source-dependent and a nonfermentablcearbonsource
Summary
Firmed with the DNA sequencing system model 373A (Applied Bio- Mapping of cis-Acting Elements within Promoter Region of systems Inc., Foster City, CA). Its direction, mutant promoters showed the same activity as Analysis of -556/-361 Fragment Involving UASl and URS the full TDH3 promoter on a fermentable carbon source but (UASIIURS) Region-Preliminary studies indicated that the only one-third of the activity on a nonfermentable carbon. TCACCCAGACACC-3', bottom strand) of the TDH3 pro- independent of carbon source, indicating that GRFlacts asa moter, which is adjacent upstream of the -4941-393 fragment trans-acting protein of the TDH3 promoter and that the containing the UASl and the URS (see Fig. 4, panel; A ). The -4941-426 fragment, which includes two sets of the UAS1-derived 13-bp elements but not the GRF1-binding site, is sufficient for the full activation of TDH3 promoter on a fermentable carbon source (see Fig. 3).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
