RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae

Abstract

We identified and mapped RNA-binding sites of yeast Saccharomyces cerevisiae translation initiation factor eIF4G1 and examined their importance for eIF4G1 function in vitro and in vivo. Yeast eIF4G1 binds to single-stranded RNA with three different sites, the regions of amino acids 1–82 (N terminus), 492–539 (middle), and 883–952 (C terminus). The middle and C-terminal RNA-binding sites represent RS (arginine and serine)-rich domains; the N-terminal site is asparagine-, glutamine- and glycine-rich. The three RNA-binding sites have similar affinity for single-stranded RNA, whereas the affinity for single-stranded RNA full-length eIF4G1 is about 100-fold higher (approximate K_d of 5 × 10⁻⁸ M). Replacement of the arginine residues in the middle RS site by alanine residues abolishes its RNA-binding activity. Deletion of individual RNA-binding sites shows that eIF4G1 molecules lacking one binding site are still active in supporting growth of yeast cells and translation in vitro, whereas eIF4G1 molecules lacking two or all three RNA-binding sites are strongly impaired or inactive. These data suggest that RNA-binding activity is required for eIF4G1 function.

Keywords

• Cell-free translation
• initiation of translation
• RNA binding
• RS domain
• yeast
• 5-FOA, fluoro-orotic acid
• Ac, acetate
• eIF, eukaryotic initiation factor
• EMC, encephalomyocarditis
• FPLC, fast performance liquid chromatography
• GSH, glutathione
• GST, glutathione-S-transferase
• IRES, internal ribosome entry site
• PBS, phosphate-buffered saline
• SDS, sodium dodecyl sulfate
• TBS, tris-buffered saline
• UTR, untranslated region