DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.     

Role of yeast elongation factor 3 in the elongation cycle

Investigation of the role of the polypeptide chain elongation factor 3 (EF-3) of yeast indicates that EF-3 participates in the elongation cycle by stimulating the function of EF-1 alpha in binding aminoacyl-tRNA (aa-tRNA) to the ribosome. In the yeast system, the binding of the ternary complex of EF-1 alpha.GTP.aa-tRNA to the ribosome is stoichiometric to the amount of EF-1 alpha. In the presence of EF-3, EF-1 alpha functions catalytically in the above mentioned reaction. The EF-3 effect is manifest in the presence of ATP, GTP, or ITP. A nonhydrolyzable analog of ATP does not replace ATP in this reaction, indicating a role of ATP hydrolysis in EF-3 function. The stimulatory effect of EF-3 is, in many respects, distinct from that of EF-1 beta. Factor 3 does not stimulate the formation of a binary complex between EF-1 alpha and GTP, nor does it stimulate the exchange of EF-1 alpha-bound GDP with free GTP. The formation of a ternary complex between EF-1 alpha.GTP.aa-tRNA is also not affected by EF-3. It appears that the only reaction of the elongation cycle that is stimulated by EF-3 is EF-1 alpha-dependent binding of aa-tRNA to the ribosome. Purified elongation factor 3, isolated from a temperature-sensitive mutant, failed to stimulate this reaction after exposure to a nonpermissive temperature. A heterologous combination of ribosomal subunits from yeast and wheat germ manifest the requirement for EF-3, dependent upon the source of the "40 S" ribosomal subunit. A combination of 40 S subunits from yeast and "60 S" from wheat germ showed the stimulatory effect of EF-3 in polyphenylalanine synthesis (Chakraburtty, K., and Kamath, A. (1988) Int. J. Biochem. 20, 581-590). However, we failed to demonstrate the effect of EF-3 in binding aa-tRNA to such a heterologous combination of the ribosomal subunits.     

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.     

Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity.

The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus.     

A general upstream binding factor for genes of the yeast translational apparatus.

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.     

Molecular cloning of some components of the translation apparatus of fission yeast Schizosaccharomyces pombe and a list of its cytoplasm ic proteins genes]

Full-length cDNAs of four new genes encoding cytoplasmic ribosomal proteins L14 and L20 (large ribosomal subunit) and S1 and S27 (small ribosomal subunit) were isolated and sequenced during the analysis of the fission yeast Schizosaccharomyces pombe genome. One of the Sz. pombe genes encoding translation elongation factor EF-2 was also cloned and its precise position on chromosome I established. A unified nomenclature was proposed, and the list of all known genetic determinants encoding cytoplasmic ribosomal proteins of Sz. pombe was compiled. By now, 76 genes/cDNAs encoding different ribosomal proteins have been identified in the fission yeast genome. Among them, 35 genes are duplicated and three homologous genes are identified for each of the ribosomal proteins L2, L16, P1, and P2.     

Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

Identification of two genes coding for the translation elongation factor EF-1 alpha of S. cerevisiae.

The translation elongation factor EF-1 alpha of the yeast Saccharomyces cerevisiae is coded for by two genes, called TEF1 and TEF2. Both genes were cloned. TEF1 maps on chromosome II close to LYS2. The location of TEF2 is unknown. TEF2 alone is sufficient to promote growth of the cells as shown with a strain deleted for TEF1. TEF1 and TEF2 were originally identified as two strongly transcribed genes, which most likely code for an identical or nearly identical protein as judged from S1 nuclease protection experiments with mRNA-DNA hybrids. The DNA sequence analysis of TEF1 allowed the prediction of the protein sequence. This was shown, by a search in the Dayhoff protein data bank, to represent the translation elongation factor EF-1 alpha due to the striking similarity to EF-1 alpha from the shrimp Artemia. A search for TEF1 homologous sequences in several yeast species shows, in most cases, duplicated genes and a much higher sequence conservation than among genes encoding amino acid biosynthetic enzymes.     

Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF-3). Using a rapid affinity chromatography protocol, EF-3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF-3 also showed cross-reactivity with EF-3 from S. cerevisiae. Similarly, the S. cerevisiae TEF3 gene (encoding EF-3) showed cross-hybridization with genomic DNA from C. albicans in Southern hybridization analysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction-amplified DNA fragment to screen to C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF-3 gene and its S. cerevisiae counterpart over the encoded 65-amino-acid stretch. That the cloned C. albicans sequence did indeed encode EF-3 was confirmed by demonstrating its ability to rescue an otherwise non-viable S. cerevisiae tef3:HIS3 null mutant. Thus EF-3 from C. albicans shows both structural and functional similarity to EF-3 from S. cerevisiae.     

The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3.

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.     

Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe orthologs of the peptidyl proyl isomerases that activate PP2A, reveals a role for Ypa2p in the regulation of cytokinesis

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis. Cdc7p is the first kinase in the core SIN; we have screened genetically for SIN regulators by isolating cold-sensitive suppressors of cdc7-24. Our screen yielded a mutant in SPAC1782.05, one of the two fission yeast orthologs of mammalian phosphotyrosyl phosphatase activator. We have characterized this gene and its ortholog SPAC4F10.04, which we have named ypa2 and ypa1, respectively. We find that Ypa2p is the major form of protein phosphatase type 2A activator in S. pombe. A double ypa1-Δ ypa2-Δ null mutant is inviable, indicating that the two gene products have at least one essential overlapping function. Individually, the ypa1 and ypa2 genes are essential for survival only at low temperatures. The ypa2-Δ mutant divides at a reduced cell size and displays aberrant cell morphology and cytokinesis. Genetic analysis implicates Ypa2p as an inhibitor of the septation initiation network. We also isolated a cold-sensitive allele of ppa2, the major protein phosphatase type 2A catalytic subunit, implicating this enzyme as a regulator of the septation initiation network.     

Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.     

The putative GTPase encoded by MTG3 functions in a novel pathway for regulating assembly of the small subunit of yeast mitochondrial ribosomes

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes. This gene codes for a member of the YqeH family of GTPases, which we have named MTG3 in keeping with the earlier convention. Mutations in MTG3 cause the accumulation of the 15 S rRNA precursor, previously shown to have an 80-nucleotide 5' extension. Sucrose gradient sedimentation of mitochondrial ribosomes from temperature-sensitive mtg3 mutants grown at the permissive and restrictive temperatures, combined with immunobloting with subunit-specific antibodies, indicate that Mtg3p is required for assembly of the 30 S but not 54 S ribosomal subunit. The respiratory deficient growth phenotype of an mtg3 null mutant is partially rescued by overexpression of the Mrpl4p constituent located at the peptide exit site of the 54 S subunit. The rescue is accompanied by an increase in processed 15 S rRNA. This suggests that Mtg3p and Mrpl4p jointly regulate assembly of the small subunit by modulating processing of the 15 S rRNA precursor.     

Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro

We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [(3)H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.     

Yeast ribosomal protein L24 affects the kinetics of protein synthesis and ribosomal protein L39 improves translational accuracy, while mutants lacking both remain viable

Four mutant strains from Saccharomyces cerevisiae were used to study ribosome structure and function. They included a strain carrying deletions of the two genes encoding ribosomal protein L24, a strain carrying a mutation spb2 in the gene for ribosomal protein L39, a strain carrying a deletion of the gene for L39, and a mutant lacking both L24 and L39. The mutant lacking only L24 showed just 25% of the normal polyphenylalanine-synthesizing activity followed by a decrease in P-site binding, suggesting the possibility that protein L24 is involved in the kinetics of translation. Each of the two L39 mutants displayed a 4-fold increase of their error frequencies over the wild type. This was accompanied by a substantial increase in A-site binding, typical of error-prone mutants. The absence of L39 also increased sensitivity to paromomycin, decreased the ribosomal subunit ratio, and caused a cold-sensitive phenotype. Mutant cells lacking both ribosomal proteins remained viable. Their ribosomes showed reduced initial rates caused by the absence of L24 but a normal extent of polyphenylalanine synthesis and a substantial in vivo reduction in the amount of 80S ribosomes compared to wild type. Moreover, this mutant displayed decreased translational accuracy, hypersensitivity to the antibiotic paromomycin, and a cold-sensitive phenotype, all caused mainly by the deletion of L39. Protein L39 is the first protein of the 60S ribosomal subunit implicated in translational accuracy.     

Ribosomes with large synthetic N-terminal extensions of protein S15 are active in vivo

The genes for ribosomal proteins S4, S13 or S15 were fused with the gene for staphylococcal protein A, or derivatives thereof (2A'-7A'). The gene fusions were introduced into Escherichia coli strains, mutated in the corresponding ribosomal protein gene, by transformation. These mutated ribosomal proteins cause a phenotype that can be complemented. Thus, the phenotype of the transformants was tested and the ribosomal proteins were analyzed. The S4 N-terminal fusion protein severely disturbed growth of both the mutant and the wild-type strains. The S13 C-terminal fusion protein was proteolyzed close to the fusion point, giving a ribosomal protein moiety that could assemble into the ribosome normally. S15 N-terminal fusion proteins complemented a cold-sensitive strain lacking protein S15 in its ribosomes. These fused proteins were assembled into active ribosomes. The position of S15 in the 30S ribosomal subunit is well known. Therefore, in structural studies of the ribosome in vivo, the S15 fusion proteins can be used as a physical reporter for S15.     

Sequence analysis and expression of the two genes for elongation factor 1 alpha from the dimorphic yeast Candida albicans.

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1 alpha (EF-1 alpha) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libraries of C. albicans genomic DNA. Sequence analysis of the two clones showed that regions of DNA flanking the coding regions of the two genes were not homologous, verifying the presence of two genes, called TEF1 and TEF2, for EF-1 alpha in C. albicans. The coding regions of TEF1 and TEF2 differed by only five nucleotides and encoded identical EF-1 alpha proteins of 458 amino acids. Both genes were transcribed into mRNA in vivo, as shown by hybridization of oligonucleotide probes, which bound specifically to the 3' nontranslated regions of TEF1 and TEF2, respectively, to C. albicans total RNA in Northern (RNA) blot analysis. The predicted EF-1 alpha protein of C. albicans was more similar to Saccharomyces cerevisiae EF-1 alpha than to M. racemosus EF-1 alpha. Furthermore, codon bias and the promoter and termination signals of the C. albicans EF-1 alpha proteins were remarkably similar to those of S. cerevisiae EF-1 alpha. Taken together, these results suggest that C. albicans is more closely related to the ascomycete S. cerevisiae than to the zygomycete M. racemosus.