Secondary literature sources for the eIF1a domain

For each of the hand selected primary papers associated with the eIF1a domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

eIF1A augments Ago2-mediated Dicer-independent miRNA biogenesis and RNA interference.

Yi T, Arthanari H, Akabayov B, Song H, Papadopoulos E, Qi HH, Jedrychowski M, Guttler T, Guo C, Luna RE, Gygi SP, Huang SA, Wagner G
Nat Commun. 2015; 6: 7194-7194

MicroRNA (miRNA) biogenesis and miRNA-guided RNA interference (RNAi) are essential for gene expression in eukaryotes. Here we report that translation initiation factor eIF1A directly interacts with Ago2 and promotes Ago2 activities in RNAi and miR-451 biogenesis. Biochemical and NMR analyses demonstrate that eIF1A binds to the MID domain of Ago2 and this interaction does not impair translation initiation. Alanine mutation of the Ago2-facing Lys56 in eIF1A impairs RNAi activities in human cells and zebrafish. The eIF1A-Ago2 assembly facilitates Dicer-independent biogenesis of miR-451, which mediates erythrocyte maturation. Human eIF1A (heIF1A), but not heIF1A(K56A), rescues the erythrocyte maturation delay in eif1axb knockdown zebrafish. Consistently, miR-451 partly compensates erythrocyte maturation defects in zebrafish with eif1axb knockdown and eIF1A(K56A) expression, supporting a role of eIF1A in miRNA-451 biogenesis in this model. Our results suggest that eIF1A is a novel component of the Ago2-centred RNA-induced silencing complexes (RISCs) and augments Ago2-dependent RNAi and miRNA biogenesis.

Cloning and characterization of complementary DNA encoding the eukaryotic initiation factor 2-associated 67-kDa protein (p67).

Wu S, Gupta S, Chatterjee N, Hileman RE, Kinzy TG, Denslow ND, Merrick WC, Chakrabarti D, Osterman JC, Gupta NK
J Biol Chem. 1993; 268: 10796-801

The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa glycoprotein (p67) protects eIF-2 alpha-subunit from inhibitory phosphorylation by eIF-2 kinases, such as heme-regulated inhibitor and double-stranded RNA-activated inhibitor. This promotes protein synthesis in the presence of eIF-2 kinases present in animal cells (Ray, M. K., Datta, B., Chakraborty, A., Chattopadhyay, A., Meza-Keuthen, S., and Gupta, N. K. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 539-543). In this study, the primary structure of rat p67 is determined by cDNA cloning. Based on the partial amino acid sequences of overlapping tryptic and cyanogen bromide cleaved fragments, degenerate oligonucleotides were synthesized and used as primers for the polymerase chain reaction to amplify the corresponding p67 cDNA fragment from rat liver first strand cDNA. The amplified DNA was then used as a probe to screen a rat tumor hepatoma (KRC-7) cDNA library, and a positive clone covering the entire coding region was obtained. From the cDNA sequence, an open reading frame that encodes p67 as a 480-amino acid protein with a molecular mass of 53 kilodaltons was predicted for the unglycosylated protein. The cloned cDNA was further characterized by in vitro transcription-coupled translation in micrococcal nuclease-treated reticulocyte lysate. The translated product migrated similarly to p67 in SDS-polyacrylamide gel electrophoresis and was precipitated with antibodies against p67. Northern blot analysis of rat liver poly(A)+ RNA showed a single size class (approximately 2 kilobases) of mRNA. The deduced amino acid sequence of the protein showed a highly charged N-terminal region composed of two basic polylysine blocks and an acidic aspartic acid block. The protein also exhibits significant sequence identity in the N-terminal region with human eIF-2 beta-subunit.

Chromosomal localization of the HYP2-gene in Saccharomyces cerevisiae and use of pulsed-field gel electrophoresis for detection of irregular recombination events in gene disruption experiments.

Wohl T, Baur M, Friedl AA, Lottspeich F
Electrophoresis. 1992; 13: 651-3

In the hypusine-containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4-amino-2-hydroxybutyllysine). The HP has been designated as an eucaryotic translation initiation factor--eIF-5A--because of its stimulating effect in the methionyl-puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1-gene is identical to the ANB1-gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2-gene has not been elucidated. By using pulsed-field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2-gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.

The ARD1 gene of yeast functions in the switch between the mitotic cell cycle and alternative developmental pathways.

Whiteway M, Szostak JW
Cell. 1985; 43: 483-92

Mutations in the yeast gene ARD1 lead to inability to respond to alpha-factor, inability to enter stationary phase, and inability to sporulate, suggesting an important role for the ARD1 gene product in controlling the switch between the mitotic cell cycle and alternative cell fates. MATa, ard1 cells seem to be defective in the expression of all a-specific functions, whereas MAT alpha, ard1 cells respond normally to a-factor. We propose that ARD1 is required for the expression of genes involved in a-mating functions, stationary phase, and sporulation. The ARD1 gene has been cloned and sequenced; there is weak homology between the C terminus of the ARD1 protein, the C-terminal region of MAT alpha 2, and the homeo box.

Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase.

West MG, Barlowe CK, Appling DR
J Biol Chem. 1993; 268: 153-60

Saccharomyces cerevisiae possess a monofunctional, cytoplasmic NAD-dependent 5,10-methylenetetrahydrofolate (THF) dehydrogenase that converts 5,10-methylene-THF to 5,10-methenyl-THF (Barlowe, C. K., and Appling, D.R. (1990) Biochemistry 29, 7089-7094). We have now isolated the gene encoding this enzyme from a yeast genomic library using oligonucleotide probes based on internal peptide sequences from the purified protein. Nucleotide sequence analysis reveals a 320-amino acid open reading frame that contains both of the internal peptide sequences. The predicted molecular weight (36,236) is consistent with the estimated size (33,000-38,000) of the purified protein. Disruption of the chromosomal copy of the gene resulted in loss of NAD-dependent 5,10-methylene-THF dehydrogenase activity and led to a purine requirement in certain genetic backgrounds, confirming a role for this enzyme in the oxidation of cytoplasmic one-carbon units. A single gene was mapped to chromosome XI by hybridization to a yeast chromosomal blot. We propose MTD1 as the name for this gene. Northern analysis of total yeast RNA revealed a single transcript of approximately 1,100 nucleotides. Multiple transcription initiation sites were identified between 58 and 83 base pairs upstream of the start of translation. The amino acid sequences derived from the nucleic acid sequences of seven other methylene-THF dehydrogenases cloned to date have been found to be highly homologous. Although the predicted amino acid sequence of the yeast NAD-dependent enzyme shows slight homology to the other sequences, it appears to be only distantly related to the other 5,10-methylene-THF dehydrogenases.

Isolation and structure of a yeast initiator tRNAmet gene.

Venegas A, Gonzalez E, Bull P, Valenzuela P
Nucleic Acids Res. 1982; 10: 1093-6

Sixteen bacterial clones containing yeast initiator tRNAmet genes have been isolated. The size of the BamHI fragments encoding these genes ranges from 4,000 to 23,000 base pairs. The nucleotide sequence of one member of this group has been determined. It has no intervening sequences.

Molecular cloning of bovine actin-like protein, actin2.

Tanaka T, Shibasaki F, Ishikawa M, Hirano N, Sakai R, Nishida J, Takenawa T, Hirai H
Biochem Biophys Res Commun. 1992; 187: 1022-8

Actins are major cytoskeletal components and highly conserved in evolution. In mammals, there are six actin isoforms, a pair of which shows at least 93% identity in the amino acid sequence. We have cloned cDNA for a bovine protein that is distantly related to members of the mammalian actin isotypes. The predicted amino acid sequence (418 residues long, calculated molecular mass 47369) shows that this protein, which we have named actin2, exhibits 36% identity to mammalian actins and 60% identity to the yeast actin-like protein, act2. We have concluded that actin2 defines a new class of mammalian actin-like proteins. It was also revealed that actin2 messenger RNA is expressed in a broad range of tissues.

Correlation between transcript profiles and fitness of deletion mutants in anaerobic chemostat cultures of Saccharomyces cerevisiae.

Tai SL, Snoek I, Luttik MA, Almering MJ, Walsh MC, Pronk JT, Daran JM
Microbiology. 2007; 153: 877-86

The applicability of transcriptomics for functional genome analysis rests on the assumption that global information on gene function can be inferred from transcriptional regulation patterns. This study investigated whether Saccharomyces cerevisiae genes that show a consistently higher transcript level under anaerobic than aerobic conditions do indeed contribute to fitness in the absence of oxygen. Tagged deletion mutants were constructed in 27 S. cerevisiae genes that showed a strong and consistent transcriptional upregulation under anaerobic conditions, irrespective of the nature of the growth-limiting nutrient (glucose, ammonia, sulfate or phosphate). Competitive anaerobic chemostat cultivation showed that only five out of the 27 mutants (eug1Delta, izh2Delta, plb2Delta, ylr413wDelta and yor012wDelta) conferred a significant disadvantage relative to a tagged reference strain. The implications of this study are that: (i) transcriptome analysis has a very limited predictive value for the contribution of individual genes to fitness under specific environmental conditions, and (ii) competitive chemostat cultivation of tagged deletion strains offers an efficient approach to select relevant leads for functional analysis studies.

Remarkable conservation of translation initiation factors: IF1/eIF1A and IF2/eIF5B are universally distributed phylogenetic markers.

Sorensen HP, Hedegaard J, Sperling-Petersen HU, Mortensen KK
IUBMB Life. 2001; 51: 321-7

Initiation of protein biosynthesis is an essential process occurring in cells throughout the three phylogenetic domains, Bacteria, Archaea, and Eucarya. IF1/eIF1A and IF2/eIF5B, two conserved translation initiation factors are involved in this important step of protein biosynthesis. The essentiality, universal distribution, conservation, and interspecies functional homology of both factors are a unique combination of properties ideal for molecular phylogenetic studies as demonstrated by the extensively compared SSU rRNAs. Here, we assess the use of IF1/eIF1A and IF2/eIF5B in universal and partial phylogenetic studies by comparison of sequence information from species within all three phylogenetic domains and among closely related strains of Haemophilus parainfluenzae. We conclude that the amino acid sequence of IF1/eIF1A-IF2/eIF5B is a universal phylogenetic marker and that the nucleotide sequence of the IF2/eIF5B G-domain is more credible than SSU rRNA for the construction of partial phylogenies among closely related species and strains. Because of these two application levels, IF1/eIF1A-IF2/eIF5B is a phylogenetic "dual level" marker.

Translation initiation factor eIF-5A expressed from either of two yeast genes or from human cDNA. Functional identity under aerobic and anaerobic conditions.

Schwelberger HG, Kang HA, Hershey JW
J Biol Chem. 1993; 268: 14018-25

Translation initiation factor eIF-5A (previously named eIF-4D) is an essential and highly conserved protein in eukaryotic cells that promotes formation of the first peptide bond. One of its lysine residues is post-translationally modified by spermidine to form hypusine, a unique residue required for eIF-5A activity. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B. The two genes are regulated reciprocally by oxygen, where under aerobic conditions TIF51A is expressed and TIF51B is repressed, and under anaerobic conditions the opposite occurs. In order to study the products of the two genes individually, yeast strains were constructed that express either TIF51A or TIF51B under control of a galactose promoter. Each gene gives rise to two isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic), both of which carry the hypusine modification. Expression of either TIF51A or TIF51B promotes growth under both aerobic and anaerobic conditions, indicating that the two gene products function indistinguishably. The human cDNA encoding eIF-5A also was expressed in yeast, and the plasmid shuffle technique was used to demonstrate that the human protein can substitute for the homologous yeast protein in vivo. These results indicate that human and yeast eIF-5A are not only conserved at the sequence level but are functionally interchangeable in vivo.

Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae.

Schnier J, Schwelberger HG, Smit-McBride Z, Kang HA, Hershey JW
Mol Cell Biol. 1991; 11: 3105-14

Translation intitiation factor eIF-5A (previously named eIF-4D) is a highly conserved protein that promotes formation of the first peptide bond. One of its lysine residues is modified by spermidine to form hypusine, a posttranslational modification unique to eIF-5A. To elucidate the function of eIF-5A and determine the role of its hypusine modification, the cDNA encoding human eIF-5A was used as a probe to identify and clone the corresponding genes from the yeast Saccharomyces cerevisiae. Two genes named TIF51A and TIF51B were cloned and sequenced. The two yeast proteins are closely related, sharing 90% sequence identity, and each is ca. 63% identical to the human protein. The purified protein expressed from the TIF51A gene substitutes for HeLa eIF-5A in the mammalian methionyl-puromycin synthesis assay. Strains lacking the A form of eIF-5A, constructed by disruption of TIF51A with LEU2, grow slowly, whereas strains lacking the B form, in which HIS3 was used to disrupt TIF51B, show no growth rate phenotype. However, strains with both TIF51A and TIF51B disrupted are not viable, indicating that eIF-5a is essential for cell growth in yeast cells. Northern (RNA) blot analysis shows two mRNA species, a larger mRNA (0.9 kb) transcribed from TIF51A and a smaller mRNA (0.8 kb) encoded by TIF51B. Under the aerobic growth conditions of this study, the 0.8-kb TIF51B transcript is not detected in the wild-type strain and is expressed only when TIF51A is disrupted. The TIF51A gene was altered by site-directed mutagenesis at the site of hypusination by changing the Lys codon to that for Arg, thereby producing a stable protein that retains the positive charge but is not modified to the hypusine derivative. The plasmid shuffle technique was used to replace the wild-type gene with the mutant form, resulting in failure of the yeast cells to grow. This result indicates that hypusine very likely is required for the vital in vivo function of eIF-5A and suggests a precise, essential role for the polyamine spermidine in cell metabolism.

A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

Schimmang T, Tollervey D, Kern H, Frank R, Hurt EC
EMBO J. 1989; 8: 4015-24

In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.

Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae.

Sasaki K, Abid MR, Miyazaki M
FEBS Lett. 1996; 384: 151-4

Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine, a modification of a specific lysine residue in the precursor of eukaryotic translation initiation factor 5A. We have purified deoxyhypusine synthase from yeast, and cloned and sequenced the corresponding gene encoding a 387-amino acid protein from Saccharomyces cerevisiae. Gene disruption experiments indicated that the deoxyhypusine synthase gene is essential for cell growth in yeast. This gene was shown to be an intron-free, single-copy gene, and its product can catalyze the synthesis of deoxyhypusine equally in two precursor forms of eIF-5A, derived from two distinct genes of yeast.

Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA(i)(Met) binding to the ribosome.

Saini AK, Nanda JS, Lorsch JR, Hinnebusch AG
Genes Dev. 2010; 24: 97-110

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA(i)(Met)) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1) and SE2, was found to stimulate recruitment of Met-tRNA(i)(Met) in the ternary complex (TC) with eIF2.GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1 and SI2, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA(i)(Met) in the P site for AUG selection.

Expression of translation initiation factor 4A from yeast and mouse in Saccharomyces cerevisiae.

Prat A, Schmid SR, Buser P, Blum S, Trachsel H, Nielsen PJ, Linder P
Biochim Biophys Acta. 1990; 1050: 140-5

The eukaryotic translation initiation factor 4A (eIF-4A) plays an important role in regulating initiation. To analyze its function in yeast, we carried out a mutational analysis of the TIF1 and TIF2 genes, which encode eIF-4A. Expression of these two yeast genes has also been investigated at the transcriptional level and it has been found that both are expressed in wild-type yeast cells. Analysis of the expression of eIF-4A-beta-galactosidase fusion proteins reveals that the TIF2 gene is more highly expressed than the TIF1 gene. Interestingly, the yeast eIF-4A protein shows a high degree of amino acid sequence similarity to the mouse homologue. However, we find that the mammalian factor does not support protein synthesis in yeast either in vivo or in vitro.

The joining of ribosomal subunits in eukaryotes requires eIF5B.

Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CU
Nature. 2000; 403: 332-5

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Isolation of the gene encoding the Drosophila melanogaster homolog of the Saccharomyces cerevisiae GCN2 eIF-2alpha kinase.

Olsen DS, Jordan B, Chen D, Wek RC, Cavener DR
Genetics. 1998; 149: 1495-509

Genomic and cDNA clones homologous to the yeast GCN2 eIF-2alpha kinase (yGCN2) were isolated from Drosophila melanogaster. The identity of the Drosophila GCN2 (dGCN2) gene is supported by the unique combination of sequence encoding a protein kinase catalytic domain and a domain homologous to histidyl-tRNA synthetase and by the ability of dGCN2 to complement a deletion mutant of the yeast GCN2 gene. Complementation of Deltagcn2 in yeast by dGCN2 depends on the presence of the critical regulatory phosphorylation site (serine 51) of eIF-2alpha. dGCN2 is composed of 10 exons encoding a protein of 1589 amino acids. dGCN2 mRNA is expressed throughout Drosophila development and is particularly abundant at the earliest stages of embryogenesis. The dGCN2 gene was cytogenetically and physically mapped to the right arm of the third chromosome at 100C3 in STS Dm2514. The discovery of GCN2 in higher eukaryotes is somewhat unexpected given the marked differences between the amino acid biosynthetic pathways of yeast vs. Drosophila and other higher eukaryotes. Despite these differences, the presence of GCN2 in Drosophila suggests at least partial conservation from yeast to multicellular organisms of the mechanisms responding to amino acid deprivation.

Isolation and characterization of the THI6 gene encoding a bifunctional thiamin-phosphate pyrophosphorylase/hydroxyethylthiazole kinase from Saccharomyces cerevisiae.

Nosaka K, Nishimura H, Kawasaki Y, Tsujihara T, Iwashima A
J Biol Chem. 1994; 269: 30510-6

Thiamin-phosphate pyrophosphorylase (TMP-PPase; EC 2.5.1.3) involved in de novo synthesis of thiamin in Saccharomyces cerevisiae is a bifunctional enzyme with 4-methyl-5-beta-hydroxyethylthiazole kinase (Th-kinase; EC 2.7.1.50) activity, which is an octamer of identical 60-kDa subunits (Kawasaki, Y. (1993) J. Bacteriol. 175, 5153-5158). Previous study demonstrated that the activities of both TMP-PPase and Th-kinase are reduced by the mutation of a single nuclear gene, designated THI6. We have cloned the THI6 gene from a yeast genomic library by functional complementation of the thi6 mutant and determined by DNA blot analysis that THI6 is located on chromosome XVI. The nucleotide sequence of the THI6 gene contained an open reading frame of 1,620 base pairs encoding a 540-amino acid polypeptide with a calculated molecular weight of 58,058, which is similar to the determined molecular mass of the purified bifunctional enzyme. Gene disruption demonstrated that the thi6 null strain is auxotrophic for thiamin, indicating that the THI6 protein is essential for thiamin synthesis in yeast. A recently isolated thi6 mutant, thi6-3, bearing a replacement of Glu370 by Lys370, showed a decrease in only Th-kinase activity, proving that the THI6 gene of S. cerevisiae encodes a structural gene of the thiamin biosynthetic bifunctional enzyme. Furthermore, complementation analysis of the thi6 null strain with the modified THI6 DNAs by a 12-nucleotide linker insertion suggested that a region from amino acids 138 to 187 and that from amino acids 370 to 453 are involved in functional domains of TMP-PPase and Th-kinase, respectively, whereas the COOH-terminal region is necessary for both enzyme activities. Strains conferring no Th-kinase but slight TMP-PPase activity could grow in medium without thiamin, suggesting that 4-methyl-5-beta-hydroxyethylthiazole is not involved in the pathway of de novo synthesis of thiamin via 4-methyl-5-beta-hydroxyethylthiazole monophosphate. Northern blot analysis demonstrated that THI6 gene expression is regulated at the mRNA level by intracellular thiamin pyrophosphate, a coenzyme form of thiamin, and that it requires the positive regulatory factors encoded by the THI2 and THI3 genes.

Characterization of translation initiation factor 5 (eIF5) from Saccharomyces cerevisiae. Functional homology with mammalian eIF5 and the effect of depletion of eIF5 on protein synthesis in vivo and in vitro.

Maiti T, Maitra U
J Biol Chem. 1997; 272: 18333-40

Eukaryotic translation initiation factor 5 (eIF5) interacts in vitro with the 40 S initiation complex (40 S.AUG.Met-tRNAf.eIF2.GTP) to mediate the hydrolysis of ribosome-bound GTP. In Saccharomyces cerevisiae, eIF5 is encoded by a single copy essential gene, TIF5, that encodes a protein of 45,346 daltons. To understand the function of eIF5 in vivo, we constructed a conditional mutant yeast strain in which a functional but a rapidly degradable form of eIF5 fusion protein was synthesized from the repressible GAL promoter. Depletion of eIF5 from this mutant yeast strain resulted in inhibition of both cell growth and the rate of in vivo protein synthesis. Analysis of the polysome profiles of eIF5-depleted cells showed greatly diminished polysomes with simultaneous increase in free ribosomes. Furthermore, lysates of cells depleted of eIF5 were dependent on exogenously added yeast eIF5 for efficient translation of mRNAs in vitro. This is the first demonstration that the TIF5 gene encodes a protein involved in initiation of translation in eukaryotic cells. Additionally, we show that rat eIF5 can functionally substitute yeast eIF5 in translation of mRNAs in vitro as well as in complementing in vivo a genetic disruption in the chromosomal copy of TIF5.

Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopy.

Li W, Hoffman DW
Protein Sci. 2001; 10: 2426-38

Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the beta barrel, aIF-1A contains an alpha-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome.

Sequence of the genes TIF1 and TIF2 from Saccharomyces cerevisiae coding for a translation initiation factor.

Linder P, Slonimski PP
Nucleic Acids Res. 1988; 16: 10359-10359

The amino acid sequence of eukaryotic translation initiation factor 1 and its similarity to yeast initiation factor SUI1.

Kasperaitis MA, Voorma HO, Thomas AA
FEBS Lett. 1995; 365: 47-50

Eukaryotic initiation factor eIF-1 was purified from rabbit reticulocytes. Amino acid sequence analysis revealed that the protein contained a blocked amino-terminus. After cleavage with the endoproteinase Asp-N, three peptides were sequenced. The obtained partial sequences were identical to sequences of SUI1ISO1, the human homologue of the yeast translation initiation factor SUI1. The SUI1 gene product was identified as a protein involved in the recognition of the protein synthesis initiation codon. A similar mode of action has been suggested for eIF-1.

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine in Saccharomyces cerevisiae.

Kang HA, Schwelberger HG, Hershey JW
J Biol Chem. 1993; 268: 14750-6

Translation initiation factor eIF-5A (formerly called eIF-4D) is a small, highly conserved protein in eukaryotic cells that undergoes a unique modification at one of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the first peptide bond. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B, and each gene gives rise to two hypusinated isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic). In order to study the structural and functional differences between the two isoforms, both isoelectric forms were purified from a yeast strain overexpressing TIF51A and were shown to stimulate identically the synthesis of methionyl-puromycin in a heterologous mammalian assay system. Pulse-chase labeling of yeast cells with [35S]methionine showed that the basic form, eIF-5Ab, is a precursor form of the acidic form, eIF-5Aa. Immunoprecipitation of 32P-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and phosphoamino acid analysis demonstrated that eIF-5Aa is generated by phosphorylation of eIF-5Ab on serine. Therefore eIF-5A undergoes two post-translational modifications, hypusination and phosphorylation, where the activity of the factor is dependent on the first but is not influenced in vitro by the second.

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation.

Hershey JW, Smit-McBride Z, Schnier J
Biochim Biophys Acta. 1990; 1050: 160-2

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.

Translation initiation factor eIF-2. Cloning and expression of the human cDNA encoding the gamma-subunit.

Gaspar NJ, Kinzy TG, Scherer BJ, Humbelin M, Hershey JW, Merrick WC
J Biol Chem. 1994; 269: 3415-22

Translation initiation factor eIF-2 is a heterotrimeric GTP-binding protein involved in the recruitment of methionyl-tRNA, to the 40 S ribosomal subunit. To complete our characterization of eIF-2, we cloned and characterized a human cDNA encoding the largest subunit, eIF-2 gamma. From limited peptide sequence data, degenerate oligo-nucleotide primers were designed to amplify a 118-base pair DNA fragment from a cDNA library. This fragment was used as a probe to screen for larger cDNAs and eventually a clone containing the complete eIF-2 gamma coding region (1416 base pairs) was identified. It encodes a 472-amino acid protein (51.8 kDa) and contains the three consensus GTP-binding elements. The protein shares strong homology to EF-Tu, GCD11 (the yeast homolog of eIF-2 gamma), and other EF-Tu-like proteins. Transfection of COS-1 cells with the cDNA results in overexpression of a 52-kDa protein which is specifically recognized by anti-eIF-2 gamma antibodies. Cross-linking experiments with diepoxybutane and trans-diaminedichloroplatinum(II) indicate that both the beta- and gamma-subunits of eIF-2 are in close proximity to methionyl-tRNAi in ternary complexes. Possession of the eIF-2 gamma cDNA will facilitate future investigations of the interactions of GTP and methionyl-tRNAi with eIF-2.

Interaction of ZPR1 with translation elongation factor-1alpha in proliferating cells.

Gangwani L, Mikrut M, Galcheva-Gargova Z, Davis RJ
J Cell Biol. 1998; 143: 1471-84

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1alpha (eEF-1alpha). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1alpha and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH2-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1alpha by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1alpha restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1alpha contributes to normal cellular proliferation.

Coupled release of eukaryotic translation initiation factors 5B and 1A from 80S ribosomes following subunit joining.

Fringer JM, Acker MG, Fekete CA, Lorsch JR, Dever TE
Mol Cell Biol. 2007; 27: 2384-97

The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

The Saccharomyces cerevisiae RPB4 gene is tightly linked to the TIF2 gene.

Foreman PK, Davis RW, Sachs AB
Nucleic Acids Res. 1991; 19: 2781-2781

Structural and functional similarities between the central eukaryotic initiation factor (eIF)4A-binding domain of mammalian eIF4G and the eIF4A-binding domain of yeast eIF4G.

Dominguez D, Kislig E, Altmann M, Trachsel H
Biochem J. 2001; 355: 223-30

The translation eukaryotic initiation factor (eIF)4G of the yeast Saccharomyces cerevisiae interacts with the RNA helicase eIF4A (a member of the DEAD-box protein family; where DEAD corresponds to Asp-Glu-Ala-Asp) through a C-terminal domain in eIF4G (amino acids 542-883). Mammalian eIF4G has two interaction domains for eIF4A, a central domain and a domain close to the C-terminus. This raises the question of whether eIF4A binding to eIF4G is conserved between yeast and mammalian cells or whether it is different. We isolated eIF4G1 mutants defective in eIF4A binding and showed that these mutants are strongly impaired in translation and growth. Extracts from mutants displaying a temperature-sensitive phenotype for growth have low in vitro translation activity, which can be restored by addition of the purified eIF4G1-eIF4E complex, but not by eIF4E alone. Analysis of mutant eIF4G(542-883) proteins defective in eIF4A binding shows that the interaction of yeast eIF4A with eIF4G1 depends on amino acid motifs that are conserved between the yeast eIF4A-binding site and the central eIF4A-binding domain of mammalian eIF4G. We show that mammalian eIF4A binds tightly to yeast eIF4G1 and, furthermore, that mutant yeast eIF4G(542-883) proteins, which do not bind yeast eIF4A, do not interact with mammalian eIF4A. Despite the conservation of the eIF4A-binding site in eIF4G and the strong sequence conservation between yeast and mammalian eIF4A (66% identity; 82% similarity at the amino acid level) mammalian eIF4A does not substitute for the yeast factor in vivo and is not functional in a yeast in vitro translation system.

Isolation, characterization and overexpression of the yeast gene, GLR1, encoding glutathione reductase.

Collinson LP, Dawes IW
Gene. 1995; 156: 123-7

Using degenerate oligodeoxyribonucleotides based on the N-terminal amino acid (aa) sequence of a yeast glutathione reductase (GR) CNBr-generated peptide fragment and a conserved C-terminal region of known GR aa sequences, the yeast gene encoding GR, GLR1, was isolated using PCR followed by screening of a yeast genomic DNA plasmid library. GLR1 encodes a 467-aa protein with a deduced M(r) of 51,545. Comparison with Escherichia coli and human GR sequences reveals 49.8% aa identity. Yeast cells transformed with a multicopy plasmid containing the genomic clone overproduced GR activity sixfold. GLR1 was found not to be an essential gene.

Function of eukaryotic translation initiation factor 1A (eIF1A) (formerly called eIF-4C) in initiation of protein synthesis.

Chaudhuri J, Si K, Maitra U
J Biol Chem. 1997; 272: 7883-91

We have used an efficient in vitro translation initiation system to show that the mammalian 17-kDa eukaryotic initiation factor, eIF1A (formerly designated eIF-4C), is essential for transfer of the initiator Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA to form the 40 S preinitiation complex (40 S.Met-tRNAf.eIF2.GTP). Furthermore, eIF1A acted catalytically in this reaction to mediate highly efficient transfer of the Met-tRNAf.eIF2.GTP ternary complex to 40 S ribosomal subunits. The 40 S complex formed was free of eIF1A indicating that its role in 40 S preinitiation complex formation is not to stabilize the binding of Met-tRNAf to 40 S ribosomes. Additionally, the eIF1A-mediated 40 S initiation complex formed in the presence of AUG codon efficiently joined 60 S ribosomal subunits in an eIF5-dependent reaction to form a functional 80 S initiation complex. In contrast to other reports, we found that eIF1A plays no role either in the subunit joining reaction or in the generation of ribosomal subunits from 80 S ribosomes. Our results indicate that the major function of eIF1A is to mediate the transfer of Met-tRNAf to 40 S ribosomal subunits to form the 40 S preinitiation complex.

Distinct functions of eukaryotic translation initiation factors eIF1A and eIF3 in the formation of the 40 S ribosomal preinitiation complex.

Chaudhuri J, Chowdhury D, Maitra U
J Biol Chem. 1999; 274: 17975-80

We have used an in vitro translation initiation assay to investigate the requirements for the efficient transfer of Met-tRNAf (as Met-tRNAf.eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA (or an AUG codon) to form the 40 S preinitiation complex. We observed that the 17-kDa initiation factor eIF1A is necessary and sufficient to mediate nearly quantitative transfer of Met-tRNAf to isolated 40 S ribosomal subunits. However, the addition of 60 S ribosomal subunits to the 40 S preinitiation complex formed under these conditions disrupted the 40 S complex resulting in dissociation of Met-tRNAf from the 40 S subunit. When the eIF1A-dependent preinitiation reaction was carried out with 40 S ribosomal subunits that had been preincubated with eIF3, the 40 S preinitiation complex formed included bound eIF3 (40 S.eIF3. Met-tRNAf.eIF2.GTP). In contrast to the complex lacking eIF3, this complex was not disrupted by the addition of 60 S ribosomal subunits. These results suggest that in vivo, both eIF1A and eIF3 are required to form a stable 40 S preinitiation complex, eIF1A catalyzing the transfer of Met-tRNAf.eIF2.GTP to 40 S subunits, and eIF3 stabilizing the resulting complex and preventing its disruption by 60 S ribosomal subunits.

Characterization of cDNAs encoding the p44 and p35 subunits of human translation initiation factor eIF3.

Block KL, Vornlocher HP, Hershey JW
J Biol Chem. 1998; 273: 31901-8

Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. It binds to 40 S ribosomal subunits resulting in dissociation of 80 S ribosomes, stabilizes initiator methionyl-tRNA binding to 40 S subunits, and is required for mRNA binding. eIF3 has an aggregate molecular mass of approximately 600 kDa and comprises at least 10 subunits. The cDNAs encoding eight of the subunits have been cloned previously (p170, p116, p110, p66, p48, p47, p40, and p36). Here we report the cloning and characterization of human cDNAs encoding two more subunits of human eIF3, namely eIF3-p44 and eIF3-p35. These proteins are immunoprecipitated by affinity-purified anti-eIF3-p170 antibodies, indicating they are components of the eIF3 complex. Far Western analysis shows that eIF3-p44 interacts strongly and specifically with the eIF3-p170 subunit, and weakly with p116/p110, p66, p40, and itself. eIF3-p44 contains an RNA recognition motif near its C terminus. Northwestern blotting shows that eIF3-p44 binds 18 S rRNA and beta-globin mRNA. Possession of cloned cDNAs encoding all 10 subunits of eIF3 provides the tools necessary to elucidate the functions of the individual subunits and the structure of the eIF3 complex.

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

Berset C, Zurbriggen A, Djafarzadeh S, Altmann M, Trachsel H
RNA. 2003; 9: 871-80

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.

The yeast homolog of mammalian ribosomal protein S30 is expressed from a duplicated gene without a ubiquitin-like protein fusion sequence. Evolutionary implications.

Baker RT, Williamson NA, Wettenhall RE
J Biol Chem. 1996; 271: 13549-55

In mammals, the 59-residue ribosomal protein S30 (rpS30) is synthesized as a fusion to a 74-residue ubiquitin-like protein, which is cleaved to yield mature rpS30. An artificial fusion of this ubiquitin-like protein to E. coli beta-galactosidase was not cleaved when expressed in yeast (Saccharomyces cerevisiae), indicating that yeast lack this cleaving activity. The yeast rpS30 homolog (yrpS30) was purified and sequenced to reveal a 63-residue protein with 61% sequence identity to mammalian rpS30. Degenerate oligonucleotides based on the yrpS30 sequence were used to isolate full-length yrpS30 cDNAs. Sequence analysis of five cDNA clones revealed that yrpS30 is not synthesized as a fusion to a ubiquitin-like protein but is extended at its N terminus by a single methionine residue. The corresponding gene was identified in the GenBankTM data base by sequence alignment and termed RPS30A. The gene consists of two exons separated by a 430-base pair intron, which contains consensus splicing elements. Exon 1 encodes the initiator methionine residue and is preceded by canonical yeast ribosomal protein gene promoter elements. Exon 2 encodes the 62-residue mature yrpS30. Genomic hybridization reveals that the RPS30A gene is duplicated. Disruption of the RPS30A gene is not lethal but confers a slow growth phenotype. Ribosomes in the mutant strains contain an authentic yrpS30 protein, indicating that a functional yrpS30 is expressed from the duplicated gene but that the reduced capacity for yrpS30 synthesis restricted the growth rate. Analysis of available DNA sequence data bases reveals that rpS30 is synthesized as a fusion to a ubiquitin-like protein in nematodes and mammals but unfused in yeast, plants, and protazoa.

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

Asano K, Vornlocher HP, Richter-Cook NJ, Merrick WC, Hinnebusch AG, Hershey JW
J Biol Chem. 1997; 272: 27042-52

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Conserved bipartite motifs in yeast eIF5 and eIF2Bepsilon, GTPase-activating and GDP-GTP exchange factors in translation initiation, mediate binding to their common substrate eIF2.

Asano K, Krishnamoorthy T, Phan L, Pavitt GD, Hinnebusch AG
EMBO J. 1999; 18: 1673-88

In the initiation phase of eukaryotic translation, eIF5 stimulates the hydrolysis of GTP bound to eIF2 in the 40S ribosomal pre-initiation complex, and the resultant GDP on eIF2 is replaced with GTP by the complex nucleotide exchange factor, eIF2B. Bipartite motifs rich in aromatic and acidic residues are conserved at the C-termini of eIF5 and the catalytic (epsilon) subunit of eIF2B. Here we show that these bipartite motifs are important for the binding of these factors, both in vitro and in vivo, to the beta subunit of their common substrate eIF2. We also find that three lysine-rich boxes in the N-terminal segment of eIF2beta mediate the binding of eIF2 to both eIF5 and eIF2B. Thus, eIF5 and eIF2Bepsilon employ the same sequence motif to facilitate interaction with the same segment of their common substrate. In agreement with this, archaea appear to lack eIF5, eIF2B and the lysine-rich binding domain for these factors in their eIF2beta homolog. The eIF5 bipartite motif is also important for its interaction with the eIF3 complex through the NIP1-encoded subunit of eIF3. Thus, the bipartite motif in eIF5 appears to be multifunctional, stimulating its recruitment to the 40S pre-initiation complex through interaction with eIF3 in addition to binding of its substrate eIF2.

Development and characterization of a reconstituted yeast translation initiation system.

Algire MA, Maag D, Savio P, Acker MG, Tarun SZ Jr, Sachs AB, Asano K, Nielsen KH, Olsen DS, Phan L, Hinnebusch AG, Lorsch JR
RNA. 2002; 8: 382-97

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.

Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining.

Acker MG, Shin BS, Dever TE, Lorsch JR
J Biol Chem. 2006; 281: 8469-75

Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S ribosomal subunit to the 40 S ribosomal subunit during translation initiation. Formation of the resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity of the factor for the complex and allowing it to dissociate. Here we present a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathway. Our data indicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translation initiation, including the eIF1- and eIF1A-dependent delivery of initiator methionyl-tRNA to the 40 S ribosomal subunit and subsequent GTP hydrolysis by eIF2. Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which has previously been shown to interact with the C terminus of eIF5B. Disruption of either isoleucine residue in the eIF1A C-terminal sequence DIDDI reduces the rate constant for GTP hydrolysis by approximately 20-fold, whereas changing the aspartic acid residues has no effect. Changing the isoleucines in the C terminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining. These data indicate that the interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly provides a checkpoint for correct complex formation, allowing full activation of GTP hydrolysis only upon formation of a properly organized 80 S initiation complex.