Polycistronic gene expression in yeast versus cryptic promoter elements

Abstract

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.

Translational Regulation of PTEN/MMAC1 Expression in Prostate Cancer

“…These observations are to + 1022), suggesting that the long 5'-UTR sequence inhibits consistent with previous studies, which showed that the long translation. These observations are consistent with the results 5'-UTR inhibits the translation of PDGF-B mRNA (4,28). derived from 293, HeLa, and H1299 cells shown in Fig.…”

“…In fact, it reduced the basal level expression lation of its downstream sequence, and the transcripts with of the second cistron. Thus, there is no detectable IRES activity short 5'-UTRs are much better templates for translation of a in the long 5'-UTR sequence of PDGF-B that mediates internal heterologous gene, consistent with previous findings (4,28). translation initiation.…”

“…These potential problems have recently been demonstrated on several previously believed-to-be cellular IRES elements. These IRES activities were shown to be due to cryptic promoters present in the 5'-UTRs (19,22,28,29) or due to differential splicing (30) which would create smaller monocistronic transcripts from the dicistronic DNAs for potential cap-dependent translation initiation of the second cistron. In one of these studies, the prevailing cellular IRES sequence of eIF4G (31,32) was shown to be an C/E BP beta transcription factor binding site (19).…”

A Small Stem Loop Element Directs Internal Initiation of the URE2 Internal Ribosome Entry Site in Saccharomyces cerevisiae

“…Of the aforementioned list of yeast cellular IRES elements, only the TIF4631 IRES has been defined by deletion analysis, but no structural data exists for the minimal IRES element (13). We consider comparisons with the HAP4, YAP1, TAF145, and TIF4631 IRES elements difficult in light of evidence suggesting that each contains cryptic promoter activity that may have been responsible for the internal initiation observed in the original papers that described these IRES elements (21)(22)(23). However, it is possible to make comparisons between the URE2 IRES and IRES elements in other eukaryotic systems.…”

Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene

“…Along this line, the presence of cryptic promoters in the genes TFIID, HAP4, and YAP1 encoding yeast transcription factors may be responsible for the expression of these proteins from dicistronic DNA constructs when encoded by the second ORF (Hecht et al 2002).…”