Polycistronic gene expression in yeast versus cryptic promoter elements.
Saccharomyces cerevisiae is a much preferred host for biotechnological applications. However, the expression of entire heterologous pathways, required for some potential products, is technically challenging in yeast. A possible tool would be polycistronic gene expression. Recent studies demonstrated that short 5' untranslated regions (5'UTRs) found upstream of certain genes support cap-independent translation in vitro. In this study 5'UTRs were used as linkers between genes in polycistronic constructs. Expression levels of genes located in the first, second and third position after a promoter were studied by replacing the respective gene by a promoterless green fluorescence protein (GFP) gene. S. cerevisiae transformed with these constructs was grown on different carbon sources and GFP expression was assayed. Our results demonstrate that (i) ribosomal read-through does not suffice for polycistronic gene expression in vivo, (ii) 5'TFIID and 5'HAP4 but not 5'L-A significantly improve the expression of a reporter gene located second in a bicistron, (iii) 5'TFIID, 5'HAP4 and 5'YAP1 but not 5'L-A can drive expression of a promoterless reporter gene, and (iv) expression driven from 5'TFIID, 5'HAP4 and 5'YAP1 is induced in the presence of raffinose or galactose but not in the presence of glucose. This implies that these elements unlike typical internal ribosome entry site-like structures contain small, potentially useful promoters which support carbon source-regulated expression.
Hecht
Katrin
K
Institute of Biotechnology, ETH-Zürich, Switzerland. hecht@biotech.biol.ethz.ch
Bailey
James E
JE
Minas
Wolfgang
W
D020121:5' Untranslated Regions; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008954:Models, Biological; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D020121:5' Untranslated Regions; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D005947:Glucose