Summary RNA metabolism plays a central role in cell growth. It is essential to regulate RNA synthesis, processing, stability and degradation. Conformational changes in RNA are key elements in regulating cellular processes. Recently, an increasing number of putative RNA helicases from different organisms ranging from Escherichia coli to humans and viruses have been identified. They are Involved in diverse cellular functions such as RNA splicing, ribosome assembly, initiation of translation, spermatogenesis, embryogenesis, and cell growth and division. Based on sequence homologies these proteins were grouped in a family, the D‐E‐A‐D box protein family (D‐E‐A‐D = Asp‐Glu‐Ala‐Asp). Some of the better characterized members have been shown to possess ATP‐binding and hydrolysing activities as well as ATP‐dependent RNA helicase activities. Most of the genes encoding such proteins have been isolated from yeast, on which we will focus in this review. From sequence data, three of the members form a subfamily, the D‐E‐A‐H subfamily.

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 TIFI 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 TIFI and TIF2 gene products.

Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)J or valine [eIF-4A(A66V)] were purified from Escherichia colt extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type e1IF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Sacharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.

Yeast Saccharomyces cerevisiae genes TIFI and TIF2 (translation initiation factor) encode a protein tentatively called translation initiation factor (Tif) due to the similarity of its amino acid sequence and its molecular weight to mammalian eukaryotic initiation factor 4A. To clarify whether Tif is involved in translation, we produced an affinitypurified anti-Tif antibody by using Tif isolated from a Tifoverproducing yeast strain as immunogen and an Escherichia coli strain expressing Tif from an expression vector to provide the extract for affinity purification of the antibody. By using chromatographic procedures and the affinity-purified anti-Tif antibody as probe to identify Tif-containing fractions, we purified Tif from wild-type yeast cells. When yeast cells containing the only TIF) gene on a plasmid under the control of the galactose-inducible CYCI-GALIO promoter were grown in medium containing glucose as the carbon source, the production of Tif was shut off and growth was arrested. Lysates made from these cells were inactive in in vitro translation.Addition of Tif to these lysates restored in vitro protein synthesis. These results show that Tif is a translation factor, the yeast homologue of mammalian translation initiation factor 4A.According to current models of translation initation in eukaryotes, initiation factors recognize and bind to the 5' terminal cap structure of mRNA and melt RNA secondary structure. Ribosomes then bind to or near the 5' end of the mRNA and reach the initiator AUG codon by scanning the mRNA in the 5' to 3' direction (for reviews, see refs.

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