Hans Trachsel

Crystal structure of the yeast eIF4A-eIF4G complex: An RNA-helicase controlled by protein–protein interactions

Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5′ end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4Gs middle domain and full-length eIF4A at 2.6-Å resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4As activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4As closed and open state provide a model for its RNA-helicase activity.

A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E

In the yeast Saccharomyces cerevisiae a small protein named p20 is found associated with translation initiation factor eIF4E, the mRNA cap‐binding protein. We demonstrate here that p20 is a repressor of cap‐dependent translation initiation. p20 shows amino acid sequence homology to a region of eIF4G, the large subunit of the cap‐binding protein complex eIF4F, which carries the binding site for eIF4E. Both, eIF4G and p20 bind to eIF4E and compete with each other for binding to eIF4E. The eIF4E–p20 complex can bind to the cap structure and inhibit cap‐dependent but not cap‐independent translation initiation: the translation of a mRNA with the 67 nucleotide Ω sequence of tobacco mosaic virus in its 5′ untranslated region (which was previously shown to render translation cap‐independent) is not inhibited by p20. Whereas the translation of the same mRNA lacking the Ω sequence is strongly inhibited by p20. Disruption of CAF20, the gene encoding p20, stimulates the growth of yeast cells, overexpression of p20 causes slower growth of yeast cells. These results show that p20 is a regulator of eIF4E activity which represses cap‐dependent initiation of translation by interfering with the interaction of eIF4E with eIF4G, e.g. the formation of the eIF4F–complex.

Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family.

BACKGROUND Translation initiation factor 4A (elF4A) is the prototype of the DEAD-box family of proteins. DEAD-box proteins are involved in a variety of cellular processes including splicing, ribosome biogenesis and RNA degradation. Energy from ATP hydrolysis is used to perform RNA unwinding during initiation of mRNA translation. The presence of elF4A is required for the 43S preinitiation complex to bind to and scan the mRNA. RESULTS We present here the crystal structure of the nucleotide-binding domain of elF4A at 2.0 A and the structures with bound adenosinediphosphate and adenosinetriphosphate at 2.2 A and 2.4 A resolution, respectively. The structure of the apo form of the enzyme has been determined by multiple isomorphous replacement. The ATPase domain contains a central seven-stranded beta sheet flanked by nine alpha helices. Despite low sequence homology to the NTPase domains of RNA and DNA helicases, the three-dimensional fold of elF4A is nearly identical to the DNA helicase PcrA of Bacillus stearothermophilus and to the RNA helicase NS3 of hepatitis C virus. CONCLUSIONS We have determined the crystal structure of the N-terminal domain of the elF4A from yeast as the first structure of a member of the DEAD-box protein family. The complex of the protein with bound ADP and ATP offers insight into the mechanism of ATP hydrolysis and the transfer of energy to unwind RNA. The identical fold of the ATPase domain of the DNA helicase PcrA of B. stearothermophilus and the RNA helicase of hepatitis C virus suggests a common fold for all ATPase domains of DExx- and DEAD-box proteins.

A Saccharomyces cerevisiae homologue of mammalian translation initiation factor 4B contributes to RNA helicase activity.

The TIF3 gene of Saccharomyces cerevisiae was cloned and sequenced. The deduced amino acid sequence shows 26% identity with the sequence of mammalian translation initiation factor eIF‐4B. The TIF3 gene is not essential for growth; however, its disruption results in a slow growth and cold‐sensitive phenotype. In vitro translation of total yeast RNA in an extract from a TIF3 gene‐disrupted strain is reduced compared with a wild‐type extract. The translational defect is more pronounced at lower temperatures and can be corrected by the addition of wild‐type extract or mammalian eIF‐4B, but not by addition of mutant extract. In vivo translation of beta‐galactosidase reporter mRNA with varying degree of RNA secondary structure in the 5′ leader region in a TIF3 gene‐disrupted strain shows preferential inhibition of translation of mRNA with more stable secondary structure. This indicates that Tif3 protein is an RNA helicase or contributes to RNA helicase activity in vivo.

mRNA cap-binding protein: cloning of the gene encoding protein synthesis initiation factor eIF-4E from Saccharomyces cerevisiae.

We have isolated genomic and cDNA clones encoding protein synthesis initiation factor eIF-4E (mRNA cap-binding protein) of the yeast Saccharomyces cerevisiae. Their identity was established by expression of a cDNA in Escherichia coli. This cDNA encodes a protein indistinguishable from purified eIF-4E in terms of molecular weight, binding to and elution from m7GDP-agarose affinity columns, and proteolytic peptide pattern. The eIF-4E gene was isolated by hybridization of cDNA to clones of a yeast genomic library. The gene lacks introns, is present in one copy per haploid genome, and encodes a protein of 213 amino acid residues. Gene disruption experiments showed that the gene is essential for growth.

The Saccharomyces cerevisiae translation initiation factor Tif3 and its mammalian homologue, eIF-4B, have RNA annealing activity.

The Saccharomyces cerevisiae TIF3 gene encodes the yeast homologue of mammalian translation initiation factor eIF‐4B. We have added six histidine residues to the C‐terminus of Tif3 protein (Tif3‐His6p) and purified the tagged protein by affinity chromatography. Tif3‐His6p stimulates translation and mRNA binding to ribosomes in a Tif3‐dependent in vitro system. Furthermore, it binds to single‐stranded RNA and catalyses the annealing of partially complementary RNA strands in vitro. In parallel experiments, RNA annealing activity could also be demonstrated for mammalian eIF‐4B. A role for Tif3/eIF‐4B and RNA annealing activity in the scanning process is proposed.

Structure of the β subunit of translational initiation factor elF-2

Summary A human liver cDNA encoding the β subunit of protein synthesis initiation factor 2 (elF-2) was isolated and sequenced. The 1416 bp cDNA encodes a protein of 333 amino acids (38,404 daltons) with characteristics that resemble authentic purified elF-2β. De novo synthesized elF-2β from cDNA transcripts incorporates into endogenous rabbit elF-2 complexes. The protein possesses putative GTP-binding sites, a zinc finger motif, and a highly charged N-terminal region composed of three basic polylysine blocks seperated by acidic domains. The polylysine blocks and the zinc finger motif suggest that elF-2β interacts with RNA. A yeast protein, Sui3, isolated as an extragenic suppressor of his4 initiation codon mutations, exhibits extensive sequence identity with human elF-2β, especially in the polylysine and zinc finger domains, thereby reinforcing the view that these elements are important for function.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system.

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells

The [URE3] phenotype in Saccharomyces cerevisiae is caused by the inactive, altered (prion) form of the Ure2 protein (Ure2p), a regulator of nitrogen catabolism. Ure2p has two functional domains: an N‐terminal domain necessary and sufficient for prion propagation and a C‐terminal domain responsible for nitrogen regulation. We show here that the mRNA encoding Ure2p possesses an IRES (internal ribosome entry site). Internal initiation leads to the synthesis of an N‐terminally truncated active form of the protein (amino acids 94–354) lacking the prion‐forming domain. Expression of the truncated Ure2p form (94–354) mediated by the IRES element cures yeast cells of the [URE3] phenotype. We assume that the balance between the full‐length and truncated (94–354) Ure2p forms plays an important role in yeast cell physiology and differentiation.

Autoregulation of the yeast lysyl-tRNA synthetase gene GCD5/KRS1 by translational and transcriptional control mechanisms

We cloned the GCD5 gene of S. cerevisiae and found it to be identical to KRS1, which encodes lysyl-tRNA synthetase (LysRS). The mutation gcd5-1 changes a conserved residue in the putative lysine-binding domain of LysRS. This leads to a defect in lysine binding and, consequently, to reduced charging of tRNA(Lys). Mutant gcd5-1 cells compensate for the defect in LysRS by increasing GCN4 expression at the translational level. GCN4 protein in turn stimulates transcription of GCD5, leading to increased LysRS activity. We propose an autoregulatory model in which uncharged tRNA(Lys) stimulates the protein kinase GCN2, a translational activator of GCN4, and thereby increases transcription of GCD5 and other genes regulated by GCN4.