Yeast prions and their prion-forming domain.
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Extrachromosomal Inheritance↗
Biomedical subjects
M F Tuite
Publications and source records attributed to M F Tuite.
The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis.
The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.
Amino Acid Sequence↗
Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI(+)] of Saccharomyces cerevisiae.
The cytoplasmic heritable determinant [PSI(+)] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI(+)] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI(+)] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are "cured" of [PSI(+)] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI(+)] determinant is not derived from the direct dissolution of self-replicating [PSI(+)] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI(+)] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI(+)] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.
Cell Division↗
The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure.
In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33). Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem. Using non-modified in vitro transcripts of the C. albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure. Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase. We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it.
Anticodon↗
Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism.
[PSI+] is a protein-based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion-like behaviour of the endogenous Sup35p protein release factor. [PSI+] strains exhibit a marked decrease in translation termination efficiency, which permits decoding of translation termination signals and, presumably, the production of abnormally extended polypeptides. We have examined whether the [PSI+]-induced expression of such an altered proteome might confer some selective growth advantage over [psi-] strains. Although otherwise isogenic [PSI+] and [psi-] strains show no difference in growth rates under normal laboratory conditions, we demonstrate that [PSI+] strains do exhibit enhanced tolerance to heat and chemical stress, compared with [psi-] strains. Moreover, we also show that the prion-like determinant [PSI+] is able to regulate translation termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of translation termination and a loss of the [PSI+] phenotype. We present a model to describe the prion-mediated regulation of translation termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi.
Fungal Proteins↗
The versatility of RNA structure and function. Jacques Monod Conference: New insights into the mechanism of mRNA translation: the significance of RNA structure, Aussois, France, 22-26 March 1999.
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Nucleic Acid Conformation↗
Selective advantages created by codon ambiguity allowed for the evolution of an alternative genetic code in Candida spp.
Several species of the genus Candida decode the standard leucine CUG codon as serine. This and other deviations from the standard genetic code in both nuclear and mitochondrial genomes invalidate the notion that the genetic code is frozen and universal and prompt the questions 'why alternative genetic codes evolved and, more importantly, how can an organism survive a genetic code change?' To address these two questions, we have attempted to reconstruct the early stages of Candida albicans CUG reassignment in the closely related yeast Saccharomyces cerevisiae. These studies suggest that this genetic code change was driven by selection using a molecular mechanism that requires CUG ambiguity. Such codon ambiguity induced a significant decrease in fitness, indicating that CUG reassignment can only be selected if it introduces an evolutionary edge to counteract the negative impact of ambiguity. We have shown that CUG ambiguity induces the expression of a novel set of stress proteins and triggers the general stress response, which, in turn, creates a competitive edge under stress conditions. In addition, CUG ambiguity in S. cerevisiae induces the expression of a number of novel phenotypes that mimic the natural resistance to stress characteristic of C. albicans. The identification of an evolutionary advantage created by CUG ambiguity is the first experimental evidence for a genetic code change driven by selection and suggests a novel role for codon reassignment in the adaptation to new ecological niches.
Adaptation, Biological↗
The C-terminus of eRF1 defines a functionally important domain for translation termination in Saccharomyces cerevisiae.
Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. By C-terminal deletion analysis of eRF1 from the yeast Saccharomyces cerevisiae, we show that the extreme C-terminus of this 437-amino-acid protein defines a functionally important domain for translation termination. A strain encoding eRF1 lacking the C-terminal 32 amino acids is not viable, whereas deletion of the C-terminal 19 amino acids is viable but shows a termination defect in vivo causing an enhancement of nonsense suppression. Using a combination of two-hybrid analysis and in vitro binding studies, we demonstrate that deletions encompassing the C-terminus of eRF1 cause a significant reduction in eRF3 binding to eRF1. All of the C-terminally truncated eRF1 still bind the ribosome, suggesting that the C-terminus does not constitute a ribosome-binding domain and eRF1 does not need to form a stable complex with eRF3 in order to bind the ribosome. These data, together with previously published data, suggest that the region between amino acids 411 and 418 of yeast eRF1 defines an essential functional domain that is part of the major site of interaction with eRF3. However, a stable eRF1:eRF3 complex does not have to be formed to maintain viability or efficient translation termination. Alignment of the seven known eukaryotic eRF1 sequences indicates that a highly conserved motif, GFGGIGG/A is present within the region of the C-terminus, although our deletion studies suggest that it is sequences C-terminal to this region that are functionally important.
Amino Acid Sequence↗
Translation termination and its regulation in eukaryotes: recent insights provided by studies in yeast.
In protein synthesis, the arrival of one or other of the three stop codons in the ribosomal A-site triggers the binding of a release factor (RF) to the ribosome and subsequent polypeptide chain release. In eukaryotes, the RF is composed of two proteins, eRF1 and eRF3. eRF1 is responsible for the hydrolysis of the peptidyl-tRNA, while eRF3 provides a GTP-dependent function, although its precise role remains to be defined. Recent findings on translation termination and its regulation from studies in the yeast Saccharomyces cerevisiae are reviewed and the potential role of eRF3 is discussed.
Animals↗
The influence of 5' codon context on translation termination in Saccharomyces cerevisiae.
Translation termination in vivo was studied in the yeast Saccharomyces cerevisiae using a translation-assay system. Codon changes that were made at position -2 relative to the stop codon, gave a 3.5-fold effect on termination in a release-factor-defective (sup45) mutant strain, in line with the effect observed in a wild-type strain. The influence of the -2 codon could be correlated to the charge of the corresponding amino acid residue in the nascent peptide; an acidic residue favoring efficient termination. Thus, the C-terminal end of the nascent peptide influences translation termination both in the bacterium Escherichia coli and to a lesser extent in the yeast S. cerevisiae. However, the sensitivity to the charge of the penultimate amino acid is reversed when the E. coli and S. cerevisiae are compared. Changing - 1 (P-site) codons in yeast gave a 10-fold difference in effect on the efficiency of termination. This effect could not be related to any property of the encoded last amino acid in the nascent peptide. Iso-codons read by the same tRNA (AAA/G, GAA/G) gave similar readthrough values. Codons for glutamine (CAA/G), glutamic acid (GAA/G) and isoleucine (AUA/C) that are read by different isoaccepting tRNAs are associated with an approximately twofold difference in each case in termination efficiency. This suggests that the P-site tRNA is able to influence termination at UGAC in yeast.
Base Sequence↗
Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein.
The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+. Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo. Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.
Chromosomes, Fungal↗
Missense translation errors in Saccharomyces cerevisiae.
We describe the development of a novel plasmid-based assay for measuring the in vivo frequency of misincorporation of amino acids into polypeptide chains in the yeast Saccharomyces cerevisiae. The assay is based upon the measurement of the catalytic activity of an active site mutant of type III chloramphenicol acetyl transferase (CATIII) expressed in S. cerevisiae. A His195(CAC)-->Tyr195(UAC) mutant of CATIII is completely inactive, but catalytic activity can be restored by misincorporation of histidine at the mutant UAC codon. The average error frequency of misincorporation of histidine at this tyrosine UAC codon in wild-type yeast strains was measured as 0. 5x10(-5) and this frequency was increased some 50-fold by growth in the presence of paromomycin, a known translational-error-inducing antibiotic. A detectable frequency of misincorporation of histidine at a mutant Ala195 GCU codon was also measured as 2x10(-5), but in contrast to the Tyr195-->His195 misincorporation event, the frequency of histidine misincorporation at Ala195 GCU was not increased by paromomycin, inferring that this error did not result from miscognate codon-anticodon interaction. The His195 to Tyr195 missense error assay was used to demonstrate increased frequencies of missense error at codon 195 in SUP44 and SUP46 mutants. These two mutants have previously been shown to exhibit a translation termination error phenotype and the sup44+ and sup46+ genes encode the yeast ribosomal proteins S4 and S9, respectively. These data represent the first accurate in vivo measurement of a specific mistranslation event in a eukaryotic cell and directly confirm that the eukaryotic ribosome plays an important role in controlling missense errors arising from non-cognate codon-anticodon interactions.
Alanine↗
Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview.
Expression of a gene can be controlled at many levels, including transcription, mRNA splicing, mRNA stability, translation and post-translational events such as protein stability and modification. The majority of studies to date have focused on transcriptional control mechanisms, but the importance of post-transcriptional mechanisms in regulating gene expression in eukaryotes is becoming increasingly clear. In this short review, selected examples of post-transcriptional gene regulatory mechanisms operating in both lower and higher eukaryotes will be used to highlight the plethora of such mechanisms already identified. The underlying theme is that post-transcriptional gene regulation relies on specific RNA-protein interactions that either result in the targeted degradation of the mRNA or prevent access of the ribosome to the translation start codon. Such interactions can occur in the 5' or 3' untranslated regions of an mRNA or within the decoded portion of the molecule. The importance of these regulatory mechanisms in a range of biological systems is also illustrated.
Animals↗
A conditional-lethal translation termination defect in a sup45 mutant of the yeast Saccharomyces cerevisiae.
Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRF1), which has a stop-codon-dependent peptidyl-release activity. Using a conditional-lethal mutant allele of SUP45 (sup45-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37 degrees C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRF1 plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.
Fungal Proteins↗
Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in yeast and mammalian tissues.
Polypeptide chain termination in eukaryotic cells is mediated in part by the release factor eRF1 (Sup45p). We have isolated and characterised cDNAs encoding this translation factor from Syrian hamster (Mesocricetus auratus) and human (Homo sapiens) Daudi cells. Comparison of the deduced amino acid sequence of these new eRF1 (Sup45p) sequences with those published for Saccharomyces cerevisiae, Arabidopsis thaliana, Xenopus laevis and human indicates a high degree of amino acid identity across a broad evolutionary range of species. Both the 5' and 3' UTRs of the mammalian eRF1 (Sup45p)-encoding cDNAs show an unusually high degree of conservation for non-coding regions. In addition, the presence of two different lengths of 3' UTR sequences in the mammalian eRF1 (Sup45p) cDNAs indicated that alternative polyadenylation sites might be used in vivo. Northern blot analysis demonstrated that eRF1 (Sup45p) transcripts of differing length, consistent with the use of alternative polyadenylation sites, were detectable in a wide range of mammalian tissues. The Xenopus, human and Syrian hamster eRF1 (Sup45p) cDNAs were shown to support the viability of a strain of S cerevisiae carrying an otherwise lethal sup45::HIS3 gene disruption indicating evolutionary conservation of function. However, the yeast strains expressing the heterogenous eRF1 (Sup45p) showed a defect in translation termination as defined by an enhancement of nonsense suppressor tRNA activity in vivo. Western blot analysis confirmed that Xenopus eRF1 (Sup45p) was primarily ribosome-associated when expressed in yeast indicating that the ribosome-binding domain of eRF1 (Sup45p) is also conserved.
Animals↗
Beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans.
We have identified a novel 399 bp repetitive DNA element (which we designate beta) 9bp upstream of a seryl-tRNA(CAG) gene in the genome of Candida albicans. There are two copies of the seryl-tRNA(CAG) gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl-tRNA(CAG) gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNA(Asp), tRNA(Ala) and tRNA(Ile). The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3' end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae. We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy-like transposable element in C. albicans that is closely associated with tRNA genes.
Base Sequence↗
The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation?
A number of yeasts of the genus Candida translate the standard leucine-CUG codon as serine. This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon. Translation of CUG as serine in these species is mediated by a novel serine-tRNA (ser-tRNACAG), which uniquely has a guanosine at position 33, 5' to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs. We propose that G-33 has two important functions: lowering the decoding efficiency of the ser-tRNACAG and preventing binding of the leucyl-tRNA synthetase. This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon. In addition, the novel ser-tRNACAG has 1-methylguanosine (m1G-37) at position 37, 3' to the anticodon, which is characteristic of leucine, but not serine tRNAs. Remarkably, m1G-37 causes leucylation of the ser-tRNACAG both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon. This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment. We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.
Adaptation, Physiological↗
Transfer RNA structural change is a key element in the reassignment of the CUG codon in Candida albicans.
The human pathogenic yeast Candida albicans and a number of other Candida species translate the standard leucine CUG codon as serine. This is the latest addition to an increasing number of alterations to the standard genetic code which invalidate the theory that the code is frozen and universal. The unexpected finding that some organisms evolved alternative genetic codes raises two important questions: how have these alternative codes evolved and what evolutionary advantages could they create to allow for their selection? To address these questions in the context of serine CUG translation in C.albicans, we have searched for unique structural features in seryl-tRNA(CAG), which translates the leucine CUG codon as serine, and attempted to reconstruct the early stages of this genetic code switch in the closely related yeast species Saccharomyces cerevisiae. We show that a purine at position 33 (G33) in the C.albicans Ser-tRNA(CAG) anticodon loop, which replaces a conserved pyrimidine found in all other tRNAs, is a key structural element in the reassignment of the CUG codon from leucine to serine in that it decreases the decoding efficiency of the tRNA, thereby allowing cells to survive low level serine CUG translation. Expression of this tRNA in S.cerevisiae induces the stress response which allows cells to acquire thermotolerance. We argue that acquisition of thermotolerance may represent a positive selection for this genetic code change by allowing yeasts to adapt to sudden changes in environmental conditions and therefore colonize new ecological niches.
Base Sequence↗