Galina Zhouravleva

TERMINATION OF TRANSLATION IN EUKARYOTES IS GOVERNED BY TWO INTERACTING POLYPEPTIDE CHAIN RELEASE FACTORS, ERF1 AND ERF3

Termination of translation in higher organisms is a GTP‐dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35‐like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C‐domain of the X.laevis eRF3 protein is highly conserved with other Sup35‐like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.

Poly(A)-Binding Protein Acts in Translation Termination via Eukaryotic Release Factor 3 Interaction and Does Not Influence [PSI+] Propagation

ABSTRACT Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach. We confirmed that a genetic interaction exists between eRF3 and Pab1p and showed that Pab1p overexpression enhances the efficiency of termination in SUP35 (eRF3) mutant and [PSI +] cells. This effect requires the interaction of Pab1p with eRF3. These data further strengthen the possibility that Pab1p has a role in coupling translation termination events with initiation of translation. Several lines of evidence indicate that Pab1p does not influence [PSI +] propagation. First, “[PSI +]-no-more” mutations do not affect eRF3-Pab1p two-hybrid interaction. Second, overexpression of PAB1 does not cure the [PSI +] phenotype or solubilize detectable amounts of eRF3. Third, prion-curing properties of overexpressed HSP104p, which is required for formation and maintenance of [PSI +], were not modified by excess Pab1p.

Eukaryotic release factors (eRFs) history

In the present review, we describe the history of the identification of the eukaryotic translation termination factors eRF1 and eRF3. As in the case of several proteins involved in general and essential processes in all cells (e.g., DNA replication, gene expression regulation…) the strategies and methodologies used to identify these release factors were first established in prokaryotes. The genetic investigations in Saccharomyces cerevisiae have made a major contribution in the field. A large amount of data have been produced, from which it was concluded that the SUP45 and SUP35 genes were controlling translation termination but were also involved in other functions important for the cell organization and the cell cycle accomplishment. This does not seem to be restricted to yeast but is also probably the case in eukaryotes in general. The biochemical studies of the proteins encoded by the higher eukaryote homologs of SUP45 and SUP35 were efficient and permitted the identification of eRF1 as being the key protein in the termination process, eRF3 having a stimulating role. Around 25 years were needed after the identification of sup45 and sup35 mutants for the characterization of their gene products as eRF1 and eRF3, respectively. It also has to be pointed out that if the results came first from bacteria, the identification of RF3 and eRF3 was made practically at the same time. Moreover, eRF1 was the first crystal structure obtained for a class‐1 release factor, the bacterial RF2 structure came later. The goal is now to understand at the molecular level the roles of both eRF1 and eRF3 in addition to their translation termination functions.

Viable nonsense mutants for the essential gene SUP45 of Saccharomyces cerevisiae

BackgroundTermination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45.ResultsWe have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.ConclusionsNonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.

Nonsense mutations in the essential gene SUP35 of Saccharomyces cerevisiae are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.

Characterization of the poly(A) binding proteins expressed during oogenesis and early development of Xenopus laevis

During vertebrate oogenesis and early embryogenesis, gene expression is governed mainly by translational control. The recruitment of Poly(A) Binding Protein (PABP) during poly(A) tail lengthening appears to be the key to translational activation during this period of development in Xenopus laevis. We showed that PABP1 and ePABP proteins are both present during oogenesis and early development. We selected ePABP as an eRF3 binding protein in a two‐hybrid screening of a X. laevis cDNA library and demonstrated that this protein is associated with translational complexes. It can complement essential functions of the yeast homologue Pab1p. We discuss specific expression patterns of the finely tuned PABP1 and ePABP proteins.

Biological roles of prion domains.

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences. In some cases, prion formation certainly represents a pathological process leading to a disease. However, there are several scenarios in which prions and other amyloids or amyloid-like aggregates are either shown or suspected to perform positive biological functions. Proven examples include self/nonself recognition, stress defense and scaffolding of other (functional) polymers. The role of prion-like phenomena in memory has been hypothesized. As an additional mechanism of heritable change, prion formation may in principle contribute to heritable variability at the population level. Moreover, it is possible that amyloid-based prions represent by-products of the transient feedback regulatory circuits, as normal cellular function of at least some prion proteins is decreased in the prion state.

Mouse GSPT2, but not GSPT1, can substitute for yeast eRF3 in vivo

Background The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2.

Inactivation of NMD increases viability of sup45 nonsense mutants in Saccharomyces cerevisiae

BackgroundThe nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.ResultsIn this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Δ double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants.ConclusionThis is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.

Effect of Charged Residues in the N-domain of Sup35 Protein on Prion [PSI+] Stability and Propagation

Background: The prion domain (PrD) of Sup35p can aggregate to form the [PSI+] prion. Results: Introduction of charged lysine residues (sup35KK) in the Sup35p PrD alters prion properties. Conclusion: Some sup35KK alleles lead to the formation of new prion variants. Significance: Establishment of molecular interactions influencing [PSI+] prion stability and maintenance is a step toward an understanding of prion folding. Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register β-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [PSI+] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles (sup35KK) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [PSI+] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [PSI+] prion. This suggests that the critical part of the parallel in-register β-structure for the studied [PSI+] prion variant lies in the first 63–69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35KK alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants.