RNA-binding activity of translation initiation factor eIF4G1 from Saccharomyces cerevisiae.
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 x 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.
Berset
Catherine
C
Institute for Biochemistry and Molecular Biology, University of Berne, CH-3012 Bern, Switzerland.
Zurbriggen
Andreas
A
Djafarzadeh
Siamak
S
Altmann
Michael
M
Trachsel
Hans
H
D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D017931:DNA Primers; D039603:Eukaryotic Initiation Factor-4G; D007700:Kinetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012331:RNA, Fungal / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D017931:DNA Primers; D039603:Eukaryotic Initiation Factor-4G; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C107740:TIF4631 protein, S cerevisiae; D011072:Poly U