TRANSLATIONAL REPRESSION BY THE HUMAN IRON-REGULATORY FACTOR (IRF) INSACCHAROMYCES-CEREVISIAE

The regulation of the synthesis of ferritin and erythroid 5-aminolevul inate synthase in mammalian cells is mediated by the interaction of th e iron regulatory factor (IRF) with a specific recognition site, the i ron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerev isiae. This comprised two components: a constitutively expressed repor ter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Indu ction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bea ring mRNAs could also be demonstrated in cell-free yeast extracts. Thi s work defines a minimal system for IRF/IRE translational regulation i n yeast that requires no additional mammalian-specific components, thu s providing direct proof that IRF functions as a translational repress or in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.