Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.
The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.
Schmid
S R
SR
Department of Microbiology, Biozentrum, Basel, Switzerland.
Linder
P
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D039601:Eukaryotic Initiation Factor-4A; D005800:Genes, Fungal; D005822:Genetic Vectors; D005838:Genotype; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010448:Peptide Initiation Factors / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D010448:Peptide Initiation Factors; D039601:Eukaryotic Initiation Factor-4A; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases