Efficiency Of Translation Termination Research Articles

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

An improved imaging system that corrects MS2-induced RNA destabilization.

The MS2 and MS2-coat protein (MS2-MCP) imaging system is widely used to study messenger RNA (mRNA) spatial distribution in living cells. Here, we report that the MS2-MCP system destabilizes some tagged mRNAs by activating the nonsense-mediated mRNA decay pathway. We introduce an improved version, which counteracts this effect by increasing the efficiency of translation termination of the tagged mRNAs. Improved versions were developed for both yeast and mammalian systems.

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

592 Ribosome profiling reveals distinct translation termination kinetics and efficiency between Fischer rat thyroid and human bronchial epidermal cells

Recognition of 3′ nucleotide context and stop codon readthrough are determined during mRNA translation elongation

The nucleotide context surrounding stop codons significantly affects the efficiency of translation termination. In eukaryotes, various 3′ contexts that are unfavorable for translation termination have been described; however, the exact molecular mechanism that mediates their effects remains unknown. In this study, we used a reconstituted mammalian translation system to examine the efficiency of stop codons in different contexts, including several previously described weak 3′ stop codon contexts. We developed an approach to estimate the level of stop codon readthrough in the absence of eukaryotic release factors (eRFs). In this system, the stop codon is recognized by the suppressor or near-cognate tRNAs. We observed that in the absence of eRFs, readthrough occurs in a 3′ nucleotide context-dependent manner, and the main factors determining readthrough efficiency were the type of stop codon and the sequence of the 3′ nucleotides. Moreover, the efficiency of translation termination in weak 3′ contexts was almost equal to that in the tested standard context. Therefore, the ability of eRFs to recognize stop codons and induce peptide release is not affected by mRNA context. We propose that ribosomes or other participants of the elongation cycle can independently recognize certain contexts and increase the readthrough of stop codons. Thus, the efficiency of translation termination is regulated by the 3′ nucleotide context following the stop codon and depends on the concentrations of eRFs and suppressor/near-cognate tRNAs.

Efficient In Vitro Full-Sense-Codons Protein Synthesis.

Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.

A Novel Connection Between [PSI+] Prion Formation and RNA Splicing in Saccharomyces cerevisiae

Relatively new studies on prions in Saccharomyces cerevisiae show that some protein prion conformations may be beneficial to cell survival in response to environmental stress. Sup35, a translation termination release factor, forms the prion [PSI+]. When [PSI+] forms, translation termination efficiency decreases, which has been shown to result in the alteration of the abundance of cellular proteins, including splicing factor abundance. Therefore, we are testing the hypothesis that [PSI+] prion formation alters splicing efficiency in yeast. First, we employed quantitative RT‐PCR and revealed that splicing efficiency improves upon [PSI+] prion formation. The proposed mechanisms for the effect of [PSI+] on protein abundance include nonsense suppression and non‐stop decay. Non‐stop decay would lead to degradation of the mRNA encoding a splicing protein and therefore would reduce splicing factor protein levels. Non‐sense suppression, a result of translation readthrough, generates a C‐terminally extended protein, which could alter the function of splicing factors, or alter protein levels. We are initiating RT‐PCR and Western Blot analyses to test the contribution of the two mechanisms on splicing factor abundance. To further test the relationship between RNA splicing and [PSI+] prion formation, we used knockout PCR to create splicing factor deletion mutations in a [psi‐] strain, as well as a weak [PSI+]SC37 strain or a strong [PSI+]Str strain. We found that the [PSI+]SC37 conformation can suppress the growth defect in a yeast strain with a deletion of the gene encoding SNU66, a component of the tri‐snRNP. Similarly, the [PSI+]SC37conformation can suppress the growth defect in a yeast strain with a deletion of the MUD2gene, which encodes a gene important for commitment complex formation. In addition, we found that [PSI+]SC37can rescue the splicing defect observed in a mud2Δstrain, consistent with the suppressive growth phenotype. Together, the results show that [PSI+] formation impacts RNA splicing in S. cerevisiae and our future work will focus on determining the mechanisms that underlie this effect.

Роль белков, взаимодействующих с факторами терминации трансляции eRF1 и eRF3, в регуляции трансляции и прионизации

The review discusses the role that proteins interacting with the translation termination factors eRF1 and eRF3 play in the control of protein synthesis and prionization. These proteins interact not only with each other, but also with many other proteins involved in controlling the efficiency of translation termination, and associate translation termination with other cell processes. The termination of translation is directly related not only to translation re-initiation and ribosome recycling, but also to mRNA stability and protein quality control. This connection is ensured by the interaction of eRF1 and eRF3 with proteins participating in various cell metabolic processes, such as mRNA transport from the nucleus into the cytoplasm (Dbp5/DDX19 and Gle1), ribosome recycling (Rli1/ABCE1), mRNA degradation (Upf proteins), and translation initiation (Pab1/PABP). In addition to genetic control, there is epigenetic control of translation termination. This mechanism is associated with prion polymerization of the Sup35 protein to form the [PSI^(+)] prion. The maintenance of the [PSI^(+)] prion, like other yeast prions, requires the operation of a system of molecular chaperones and protein sorting factors. The review considers in detail the interaction of the translation termination factors with proteins involved in various cellular processes.

Detailed Dissection and Critical Evaluation of the Pfizer/BioNTech and Moderna mRNA Vaccines.

The design of Pfizer/BioNTech and Moderna mRNA vaccines involves many different types of optimizations. Proper optimization of vaccine mRNA can reduce dosage required for each injection leading to more efficient immunization programs. The mRNA components of the vaccine need to have a 5′-UTR to load ribosomes efficiently onto the mRNA for translation initiation, optimized codon usage for efficient translation elongation, and optimal stop codon for efficient translation termination. Both 5′-UTR and the downstream 3′-UTR should be optimized for mRNA stability. The replacement of uridine by N1-methylpseudourinine () complicates some of these optimization processes because is more versatile in wobbling than U. Different optimizations can conflict with each other, and compromises would need to be made. I highlight the similarities and differences between Pfizer/BioNTech and Moderna mRNA vaccines and discuss the advantage and disadvantage of each to facilitate future vaccine improvement. In particular, I point out a few optimizations in the design of the two mRNA vaccines that have not been performed properly.

Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae.

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Poly(A)-Binding Protein Regulates the Efficiency of Translation Termination

Multiple factors influence translation termination efficiency, including nonsense codon identity and immediate context. To determine whether the relative position of a nonsense codon within an open reading frame (ORF) influences termination efficiency, we quantitate the production of prematurely terminated and/or readthrough polypeptides from 26 nonsense alleles of 3 genes expressed in yeast. The accumulation of premature termination products and the extent of readthrough for the respective premature termination codons (PTCs) manifest a marked dependence on PTC proximity to the mRNA 3' end. Premature termination products increase in relative abundance, whereas readthrough efficiencies decrease progressively across different ORFs, and readthrough efficiencies for a PTC increase in response to 3' UTR lengthening. These effects are eliminated and overall translation termination efficiency decreases considerably in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and also suggest that inefficient termination is a trigger for nonsense-mediated mRNA decay (NMD).

The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes

The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes

Impact of C-terminal amino acid composition on protein expression in bacteria.

The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.

The Influence of A/G Composition of 3' Stop Codon Contexts on Translation Termination Efficiency in Eukaryotes

Translation termination is a finishing step of protein biosynthesis. The significant role in this process belongs not only to protein factors of translation termination but also to the nearest nucleotide environment of stop codons. There are numerous descriptions of stop codons readthrough, which is due to specific nucleotide sequences behind them. However, represented data are segmental and don't explain the mechanism of the nucleotide context influence on translation termination. It is well known that stop codon UAA usage is preferential for A/T-rich genes, and UAG, UGA-for G/C-rich genes, which is related to an expression level of these genes. We investigated the connection between a frequency of nucleotides occurrence in 3' area of stop codons in the human genome and their influence on translation termination efficiency. We found that 3' context motif, which is cognate to the sequence of a stop codon, stimulates translation termination. At the same time, the nucleotide composition of 3' sequence that differs from stop codon, decreases translation termination efficiency.

MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure.

Mitochondrial dysfunction and proteostasis failure frequently coexist as hallmarks of neurodegenerative disease. How these pathologies are related is notwell understood. Here, we describe a phenomenon termed MISTERMINATE (mitochondrial-stress-induced translational termination impairment and protein carboxyl terminal extension), which mechanistically links mitochondrial dysfunction with proteostasis failure. We show that mitochondrial dysfunction impairs translational termination of nuclear-encoded mitochondrial mRNAs, including complex-I 30kD subunit (C-I30) mRNA, occurring on the mitochondrial surface in Drosophila and mammalian cells. Ribosomes stalled at the normal stop codon continue to add to the C terminus of C-I30 certain amino acids non-coded by mRNA template. C-terminally extended C-I30 is toxic when assembled into C-I and forms aggregates in the cytosol. Enhancing co-translational quality control prevents C-I30 C-terminal extension and rescues mitochondrial and neuromuscular degeneration in a Parkinson's disease model. These findings emphasize theimportance of efficient translation termination and reveal unexpected link between mitochondrial health and proteome homeostasis mediated by MISTERMINATE.

A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling.

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

The three faces of Sup35.

Sup35p is an essential protein in yeast that functions in complex with Sup45p for efficient translation termination. Although some may argue that this function is the only important attribute of Sup35p, there are two additional known facets of Sup35p's biology that may provide equally important functions for yeast; both of which involve various strategies for coping with stress. Recently, the N-terminal and middle regions (NM) of Sup35p, which are not required for translation termination function, have been found to provide stress-sensing abilities and facilitate the phase separation of Sup35p into biomolecular condensates in response to transient stress. Interestingly, the same NM domain is also required for Sup35p to misfold and enter into aggregates associated with the [PSI+ ] prion. Here, we review these three different states or "faces" of Sup35p. For each, we compare the functionality and necessity of different Sup35p domains, including the role these domains play in facilitating interactions with important protein partners, and discuss the potential ramifications that each state affords yeast cells under varying environmental conditions.

Optimizing Phage Translation Initiation

Phage as an anti-bacterial agent must be efficient in killing bacteria, and consequently needs to replicate efficiently. Protein production is a limiting step in replication in almost all forms of life, including phages. Efficient protein production depends on the efficiency of translation initiation, elongation and termination, with translation initiation often being rate limiting. Initiation signals such as Shine-Dalgarno (SD) sequences and start codon are decoded by anti-SD sequences and initiation tRNA, respectively. While the decoding machinery cannot be readily modified, the signals can be engineered to increase the efficiency of their decoding. Here I review our understanding of the translation machinery to facilitate the engineering of optimal translation initiation signals for facilitating the design of phage protein-coding genes, including 1) accurate characterization of the 3’ end of 16S rRNA by using RNA-Seq data, 2) identification of the optimal SD/aSD interaction, and 3) reduction of secondary structure in sequences flanking the start codon.

Nonsense Suppression Position Effect Implicates Poly(A)-Binding Protein in the Regulation of Translation Termination

Readthrough of translation termination codons, also known as nonsense suppression, is a relatively inefficient process mediated by ribosomal A site recognition and insertion of near-cognate tRNAs. Multiple factors influence readthrough efficiency, including nonsense codon specificity and context. To determine whether nonsense codon position in a gene influences the extent of readthrough, we generated a series of LUC nonsense alleles and quantitated both readthrough and termination efficiencies at each nonsense codon in yeast cells lacking nonsense-mediated mRNA decay (NMD) activity. Readthrough efficiency for premature termination codons (PTCs) manifested a marked dependence on PTC proximity to the mRNA 3’-end, decreasing progressively across the LUC ORF but increasing with 3’-UTR lengthening. These effects were eliminated, and translation termination efficiency decreased considerably, in cells harboring pab1 mutations. Our results support a critical role for poly(A)-binding protein in the regulation of translation termination and suggest that inefficient termination is the trigger for NMD.

Characterization of Eukaryotic Release Factor 3 (eRF3) Translation Termination Factor in Plants

Eukaryotic translation termination is mediated by two conserved interacting release factors, eRF1 and eRF3. eRF1 recognizes the stop codon and promotes the hydrolysis of the polypeptide chain, while eukaryotic eRF3 stimulates eRF1 release activity in the presence of GTP. It is widely believed that translation termination is highly conserved in eukaryotes. However, recent results that eRF1 is present in multiple, partially redundant copies in plants and that eRF1 expression is controlled by a complex, plant-specific autoregulatory circuit suggest that regulation of translation termination might be especially complex in plants. Surprisingly, very little is known about translation termination in plant, for instance, the eRF3 termination factor has not been analyzed in plants yet. Thus, we wanted to identify and characterize the eRF3 translation termination factor in plants. By combining a range of transient and transgenic assay here, we identified plant eRF3 and showed that it directly interacts with eRF1. In contrast to eRF1, plant eRF3 is not autoregulated, while eRF3 and eRF1 expressions are connected. We also demonstrated that eRF3 interacts with the core NMD factor, UPF1, and the expression of eRF3 is NMD regulated in certain plant species suggesting that in addition to the normal translation termination, eRF3 could be connected to plant nonsense-mediated decay (NMD). Finally, it appears that the plant termination factors are present in physiologically different concentrations, while eRF1 concentration limits the efficiency of both translation termination and NMD, eRF3 is present in non-limiting concentration.