Isolation and characterization of the structural gene encoding elongation factor 3

The unique yeast translational factor EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA to the ribosome. We have isolated the structural gene encoding EF-3 from the yeast Saccharomyces cerevisiae. The YEF3 gene is found in one copy per haploid genome and is essential for vegetative growth. DNA sequence analysis reveals that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence has two repeats of a nucleotide-binding motif. Each of these repeats shows similarity to the nucleotide-binding motif of hydrophilic, membrane-associated ATPases including human multidrug resistant protein MDR. Factor 3 manifests ribosome-dependent ATP hydrolysis. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity and EF-3 protein levels by 3-5-fold. Yeast strains containing elevated EF-3 protein levels also exhibit increased sensitivity to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. These observations suggest that EF-3 may affect translational accuracy.     

Functional interaction of yeast elongation factor 3 with yeast ribosomes

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.     

Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling.

The elongation factor 2 (EF-2) genes of the yeast Saccharomyces cerevisiae have been cloned and characterized with the ultimate goal of gaining a better understanding of the mechanism and control of protein synthesis. Two genes (EFT1 and EFT2) were isolated by screening a bacteriophage lambda yeast genomic DNA library with an oligonucleotide probe complementary to the domain of EF-2 that contains diphthamide, the unique posttranslationally modified histidine that is specifically ADP-ribosylated by diphtheria toxin. Although EFT1 and EFT2 are located on separate chromosomes, the DNA sequences of the two genes differ at only four positions out of 2526 base pairs, and the predicted protein sequences are identical. Genetic deletion of each gene revealed that at least one functional copy of either EFT gene is required for cell viability. Messenger RNA levels of yeast EF-2 parallel cellular growth and peak in mid-log phase cultures. The EF-2 protein sequence is strikingly conserved through evolution. Yeast EF-2 is 66% identical to, and shares over 85% homology with, human EF-2. In addition, yeast and mammalian EF-2 share identical sequences at two critical functional sites: (i) the domain containing the histidine residue that is modified to diphthamide and (ii) the threonine residue that is specifically phosphorylated in vivo in mammalian cells by calmodulin-dependent protein kinase III, also known as EF-2 kinase. Furthermore, yeast EF-2 also contains the Glu-X-X-Arg-X-Ile-Thr-Ile "effector" sequence motif that is conserved among all known elongation factors, and its GTP-binding domain exhibits strong homology to the G-domain of Escherichia coli elongation factor Tu (EF-Tu) and other G-protein family members. Based upon these observations, we have modeled the G-domain of the deduced EF-2 protein sequence to the solved crystallographic structure for EF-Tu.     

Translation elongation factor-3 (EF-3): An evolving eukaryotic ribosomal protein?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome but are nevertheless essential for translation elongation in vivo. The EF-3 polypeptide has been identified in a wide range of fungal species and the gene encoding EF-3 (YEF3) has been isolated from four fungal species (Saccharomyces cerevisiae, Candida albicans, Candida guillermondii, andPneumocystis carinii). Computer-assisted analysis of the predictedS. cerevisiae EF-3 amino acid sequence was used to identify several potential functional domains; two ATP binding/catalytic domains conserved with equivalent domains in members of the ATP-Binding Cassette (ABC) family of proteins, an aminoterminal region showing significant similarity to theE. coli S5 ribosomal protein, and regions of predicted interaction with rRNA, tRNA, and mRNA. Furthermore, EF-3 was also found to display amino acid similarity to myosin proteins whose cellular function is to provide the motive force of muscle. The identification of these regions provides clues to both the evolution and function of EF-3. The predicted functional regions are conserved among all known fungal EF-3 proteins and a recently described homologue encoded by the Chlorella virus CVK2. We propose that EF-3 may play a role in the ribosomal optimization of the accuracy of fungal protein synthesis by altering the conformation and activity of a ribosomal accuracy center, which is equivalent to the S4-S5-S12 ribosomal protein accuracy center domain of theE. coli ribosome. Furthermore, we suggest that EF-3 represents an evolving ribosomal protein with properties analogous to the intrinsic ATPase activities of higher eukaryotic ribosomes, which has wider implications for the evolutionary divergence of fungi from other eukaryotes. Correspondence to: M.F. Tuite     

Mutations in Elongation Factor 1β, a Guanine Nucleotide Exchange Factor, Enhance Translational Fidelity

Translation elongation factor 1beta (EF-1beta) is a member of the family of guanine nucleotide exchange factors, proteins whose activities are important for the regulation of G proteins critical to many cellular processes. EF-1beta is a highly conserved protein that catalyzes the exchange of bound GDP for GTP on EF-1alpha, a required step to ensure continued protein synthesis. In this work, we demonstrate that the highly conserved C-terminal region of Saccharomyces cerevisiae EF-1beta is sufficient for normal cell growth. This region of yeast and metazoan EF-1beta and the metazoan EF-1beta-like protein EF-1delta is highly conserved. Human EF-1beta, but not human EF-1delta, is functional in place of yeast EF-1beta, even though both EF-1beta and EF-1delta have previously been shown to have guanine nucleotide exchange activity in vitro. Based on the sequence and functional homology, mutagenesis of two C-terminal residues identical in all EF-1beta protein sequences was performed, resulting in mutants with growth defects and sensitivity to translation inhibitors. These mutants also enhance translational fidelity at nonsense codons, which correlates with a reduction in total protein synthesis. These results indicate the critical function of EF-1beta in regulating EF-1alpha activity, cell growth, translation rates, and translational fidelity.     

Identification of a ribosomal ATPase in Escherichia coli cells

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.     

Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli

The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone. Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains. Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response. The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction.     

Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.     

The mitotic apparatus-associated 51-kDa protein from sea urchin eggs is a GTP-binding protein and is immunologically related to yeast polypeptide elongation factor 1 alpha

We investigated the biochemical characteristics of the 51-kDa protein that is a major mitotic apparatus-associated basic protein of sea urchin eggs (Toriyama, M., Ohta, K., Endo, S., and Sakai, H. (1988) Cell Motil. Cytoskeleton 9, 117-128). The amino acid composition of the 51-kDa protein was apparently different from those of tubulin, actin, histones, and myelin basic protein; yet it was similar to those of polypeptide elongation factors 1 alpha (EF-1 alpha). In addition, antibody to EF-1 alpha from yeast cross-reacted with the 51-kDa protein. [3H] GTP binding activity was detected in the phosphocellulose-purified fraction (PC fraction) which predominantly contained the 51-kDa protein and was shown to be specific to GTP, GDP, guanylyl imidodiphosphate, and ITP. Photo-affinity labeling using [alpha-32P]8-azidoguanosine triphosphate (8-azido-GTP) demonstrated that a 51-kDa polypeptide in the PC fraction specifically bound 8-azido-GTP. This GTP-binding polypeptide was bound to a GTP affinity column, could be eluted by the addition of GTP, and was immunoreactive with anti-51-kDa protein antibodies. When the PC fraction was applied to a gel filtration chromatography column, GTP binding activity was completely coeluted with the 51-kDa protein. Furthermore, the PC fraction and the gel filtration-purified fraction had EF-1 alpha activity: [14C]Phe-tRNA transferring activity to ribosomes in the presence of poly(U) and ribosome-dependent GTPase activity. The results indicate that the mitotic apparatus-associated 51-kDa protein is a GTP-binding protein and suggest that it is structurally and functionally related to yeast EF-1 alpha.     

Construction of a Sorbitol-Based Vector for Expression of Heterologous Proteins in Saccharomyces cerevisiae

A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.     

Functional expression of hexahistidine-tagged beta-subunit of yeast F1-ATPase and isolation of the enzyme by immobilized metal affinity chromatography

Mitochondrial ATP synthase (F1Fo-ATPase) catalyzes the terminal step of oxidative phosphorylation. In this paper, we demonstrate the functional expression of the hexahistidine-tagged beta-subunit of yeast ATP synthase and the purification of the F1-ATPase from yeast cells. A gene encoding the beta-subunit from Saccharomyces cerevisiae was modified to encode a protein of which the original N-terminus import signal sequence was replaced by a sequence containing the import signal sequence of a mitochondrial ATPase inhibitor, its processing site, and six consecutive histidines. Expression of the modified gene generated a functional F1Fo complex in host yeast cells lacking a functional copy of the endogenous ATP2 gene, as judged by growth of rescued cells on lactate medium. F1 was extracted from the yeast mitochondria by chloroform treatment and purified by immobilized metal affinity chromatography and gel filtration chromatography. The specific activity of the purified F1 was comparable to that of the wild-type enzyme, and the F1 contained all of the 5 known subunits (alpha, beta, gamma, delta, and epsilon). Moreover, the activity of the F1 was completely inhibited by the specific ATPase inhibitor protein, IF1. These results indicate that F1 containing the tagged beta-subunit is fully assembled and active. The application of this novel procedure simplifies the number of steps required for the isolation of F1 used for studying the molecular mechanism of catalysis and regulation of the enzyme.     

Effect of cyclic AMP and cyclic GMP on the autophosphorylation of elongation factor 1 from wheat embryos

Extensively purified EF-1H (EF-1 alpha beta beta' gamma) from wheat embryos had a protein kinase activity and phosphorylated EF-1 beta which is one of the two EF-Ts-like factors (EF-1 beta and EF-1 beta'). In this reaction ATP and GTP were equally effective as phosphate donors, and threonine residue was phosphorylated. At 10(-7)M, 3', 5' cyclic GMP stimulated both the phosphorylation and phe-tRNA binding reactions, whereas 3', 5' cyclic AMP inhibited both reactions. These findings indicate that phosphorylation of EF-1H may play an important role in the translational control of protein biosynthesis at the elongation step.     

Protein folding within the cell is influenced by controlled rates of polypeptide elongation.

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.     

Characterization of the ATPase and GTPase activities of elongation factor 3 (EF-3) purified from yeasts

Three steps of chromatography of a post-ribosomal supernatant fraction have provided a highly purified preparation of peptide elongation factor 3 (EF-3) with a molecular weight of 125,000 from the typical budding yeast Saccharomyces carlsbergensis and of the factor with a molecular weight of 120,000 from the fission yeast Schizosaccharomyces pombe. Both of the proteins consist of a single peptide chain. The purified factors fulfilled the requirement for polyphenylalanine synthesis on yeast ribosomes and exhibited strong ATPase and GTPase activities dependent on yeast ribosomes. The activity profiles of the nucleotidases dependent on pH and salt concentration and the inhibition studies indicated that the ATPase and GTPase activities of EF-3 were displayed by the same active site with a wide substrate specificity, showing the highest activity with ATP. Those experiments also revealed that the ATPase and GTPase of EF-3 were characteristically different from the GTPases of EF-1 alpha and EF-2. Both Km and kcat of EF-3 for ATP (Km = 0.12 mM and Kcat = 610 mol/mol/min) and GTP (Km = 0.20 mM and kcat = 390 mol/mol/min) are much higher than those of the GTPases of EF-1 alpha and EF-2. Inactivation experiments and studies on the ATP effect led us to conclude that this ATPase activity was an essential requirement for the functional role of EF-3 and therefore, in addition to the GTPases of EF-1 alpha and EF-2, the third nucleoside triphosphate hydrolyzing step by the ATPase of EF-3 was necessary for the yeast peptide elongation cycle.     

Eukaryotic protein elongation factors

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with [alpha-32P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.     

Studies on the mode of action of hygromycin B, an inhibitor of translocation in eukaryotes.

Hygromycin B is an unusual aminoglycoside antibiotic active against both prokaryotic and eukaryotic cells. Hygromycin B at 0.38 mM concentration completely halts yeast cell growth in rich media, presumably by preventing protein synthesis by cytoplasmic ribosomes. Polypeptide synthesis in cell-free extracts from rabbit reticulocytes, wheat germ and yeast is strongly blocked by low concentrations of hygromycin B. The antibiotic inhibits peptide chain elongation by yeast polysomes by preventing elongation factor EF-2-dependent translocation, although it does not affect either the formation of the EF-2-GTP-ribosome complex or the EF-2- and ribosome-dependent GTP hydrolysis which takes place uncoupled from translocation. The inhibition of translocation by hygromycin B might result from the stabilization of peptidyl-tRNA bound to the ribosomal acceptor site, since the stability of [3H]Phe-tRNA-EF-1-poly(U)-ribosome and [3H]Phe-tRNA-poly(U)-ribosome complexes is increased in the presence of hygromycin B. The inhibition of polyphenylalanine synthesis by reticulocyte ribosomes and enzymic translocation of peptidyl-tRNA by yeast polysomes can be reversed by increasing concentrations of EF-2 suggesting a relationship between the binding sites of EF-2 and hygromycin B on the ribosome. Neither non-enzymic translocation, that takes place in the presence of high potassium concentrations, nor the peptide bondforming step are affected by hygromycin B.