Translational regulation enhances distinction of cell types in the nervous system.

Multicellular organisms are composed of specialized cell types with distinct proteomes. While recent advances in single-cell transcriptome analyses have revealed differential expression of mRNAs, cellular diversity in translational profiles remains underinvestigated. By performing RNA-seq and Ribo-seq in genetically defined cells in the Drosophila brain, we here revealed substantial post-transcriptional regulations that augment the cell-type distinctions at the level of protein expression. Specifically, we found that translational efficiency of proteins fundamental to neuronal functions, such as ion channels and neurotransmitter receptors, was maintained low in glia, leading to their preferential translation in neurons. Notably, distribution of ribosome footprints on these mRNAs exhibited a remarkable bias toward the 5' leaders in glia. Using transgenic reporter strains, we provide evidence that the small upstream open-reading frames in the 5' leader confer selective translational suppression in glia. Overall, these findings underscore the profound impact of translational regulation in shaping the proteomics for cell-type distinction and provide new insights into the molecular mechanisms driving cell-type diversity.

Bacterial Ribosomes Induce Plasticity in Mouse Adult Fibroblasts.

The incorporation of bacterial ribosome has been reported to induce multipotency in somatic and cancer cells which leads to the conversion of cell lineages. Queried on its universality, we observed that bacterial ribosome incorporation into trypsinized mouse adult fibroblast cells (MAF) led to the formation of ribosome-induced cell clusters (RICs) that showed strong positive alkaline phosphatase staining. Under in vitro differentiation conditions, RICs-MAF were differentiated into adipocytes, osteoblasts, and chondrocytes. In addition, RICs-MAF were able to differentiate into neural cells. Furthermore, RICs-MAF expressed early senescence markers without cell death. Strikingly, no noticeable expression of renowned stemness markers like Oct4, Nanog, Sox2, etc. was observed here. Later RNA-sequencing data revealed the expression of rare pluripotency-associated markers, i.e., Dnmt3l, Sox5, Tbx3 and Cdc73 in RICs-MAF and the enrichment of endogenous ribosomal status. These observations suggested that RICs-MAF might have experienced a non-canonical multipotent state during lineage conversion. In sum, we report a unique approach of an exo-ribosome-mediated plastic state of MAF that is amenable to multi-lineage conversion.

Unveiling the translational dynamics of lychee (Litchi chinesis Sonn.) in response to cold stress.

Cold stress poses a significant threat to the quality and productivity of lychee (Litchi chinensis Sonn.). While previous research has extensively explored the genomic and transcriptomic responses to cold stress in lychee, the translatome has not been thoroughly investigated. This study delves into the translatomic landscape of the 'Xiangjinfeng' cultivar under both control and low-temperature conditions using RNA sequencing and ribosome profiling. We uncovered a significant divergence between the transcriptomic and translatomic responses to cold exposure. Additionally, bioinformatics analyses underscored the crucial role of codon occupancy in lychee's cold tolerance mechanisms. Our findings reveal that the modulation of translation via codon occupancy is a vital strategy to abiotic stress. Specifically, the study identifies ribosome stalling, particularly at the E site AAU codon, as a key element of the translation machinery in lychee's response to cold stress. This work enhances our understanding of the molecular dynamics of lychee's reaction to cold stress and emphasizes the essential role of translational regulation in the plant's environmental adaptability.

Perturbation of METTL1-mediated tRNA N7- methylguanosine modification induces senescence and aging.

Cellular senescence is characterized by a decrease in protein synthesis, although the underlying processes are mostly unclear. Chemical modifications to transfer RNAs (tRNAs) frequently influence tRNA activity, which is crucial for translation. We describe how tRNA N7-methylguanosine (m7G46) methylation, catalyzed by METTL1-WDR4, regulates translation and influences senescence phenotypes. Mettl1/Wdr4 and m7G gradually diminish with senescence and aging. A decrease in METTL1 causes a reduction in tRNAs, especially those with the m7G modification, via the rapid tRNA degradation (RTD) pathway. The decreases cause ribosomes to stall at certain codons, impeding the translation of mRNA that is essential in pathways such as Wnt signaling and ribosome biogenesis. Furthermore, chronic ribosome stalling stimulates the ribotoxic and integrative stress responses, which induce senescence-associated secretory phenotype. Moreover, restoring eEF1A protein mitigates senescence phenotypes caused by METTL1 deficiency by reducing RTD. Our findings demonstrate that tRNA m7G modification is essential for preventing premature senescence and aging by enabling efficient mRNA translation.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Multiscale modeling uncovers 7q11.23 copy number variation-dependent changes in ribosomal biogenesis and neuronal maturation and excitability.

Copy number variation (CNV) at 7q11.23 causes Williams-Beuren syndrome (WBS) and 7q microduplication syndrome (7Dup), neurodevelopmental disorders (NDDs) featuring intellectual disability accompanied by symmetrically opposite neurocognitive features. Although significant progress has been made in understanding the molecular mechanisms underlying 7q11.23-related pathophysiology, the propagation of CNV dosage across gene expression layers and their interplay remains elusive. Here we uncovered 7q11.23 dosage-dependent symmetrically opposite dynamics in neuronal differentiation and intrinsic excitability. By integrating transcriptomics, translatomics, and proteomics of patient-derived and isogenic induced neurons, we found that genes related to neuronal transmission follow 7q11.23 dosage and are transcriptionally controlled, while translational factors and ribosomal genes are posttranscriptionally buffered. Consistently, we found phosphorylated RPS6 (p-RPS6) downregulated in WBS and upregulated in 7Dup. Surprisingly, p-4EBP was changed in the opposite direction, reflecting dosage-specific changes in total 4EBP levels. This highlights different dosage-sensitive dyregulations of the mTOR pathway as well as distinct roles of p-RPS6 and p-4EBP during neurogenesis. Our work demonstrates the importance of multiscale disease modeling across molecular and functional layers, uncovers the pathophysiological relevance of ribosomal biogenesis in a paradigmatic pair of NDDs, and uncouples the roles of p-RPS6 and p-4EBP as mechanistically actionable relays in NDDs.

SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression.

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed "stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes" (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper's transparent peer review process is included in the supplemental information.

The cytidine deaminase APOBEC3A regulates nucleolar function to promote cell growth and ribosome biogenesis.

Cancer initiates as a consequence of genomic mutations and its subsequent progression relies in part on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. In an attempt to form a connection between these 2 cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratory's established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. Through siRNA depletion experiments, we highlight APOBEC3A's requirement in making ribosomes and specific role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. We demonstrate that a subset of APOBEC3A resides within the nucleolus and associates with critical ribosome biogenesis factors. Mechanistic insight was revealed by transient overexpression of both wild-type and a catalytically dead mutated APOBEC3A, which both increase cell growth and protein synthesis. Through an innovative nuclear RNA sequencing methodology, we identify only modest predicted APOBEC3A C-to-U target sites on the pre-rRNA and pre-mRNAs. Our work reveals a potential direct role for APOBEC3A in ribosome biogenesis likely independent of its editing function. More broadly, we found an additional function of APOBEC3A in cancer pathology through its function in ribosome biogenesis, expanding its relevance as a target for cancer therapeutics.

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre.

Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the 'critical region' of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of ß-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

The orchestration of cell-cycle reentry and ribosome biogenesis network is critical for cardiac repair.

Rationale: Myocardial infarction (MI) is a severe global clinical condition with widespread prevalence. The adult mammalian heart's limited capacity to generate new cardiomyocytes (CMs) in response to injury remains a primary obstacle in developing effective therapies. Current approaches focus on inducing the proliferation of existing CMs through cell-cycle reentry. However, this method primarily elevates cyclin dependent kinase 6 (CDK6) and DNA content, lacking proper cytokinesis and resulting in the formation of dysfunctional binucleated CMs. Cytokinesis is dependent on ribosome biogenesis (Ribo-bio), a crucial process modulated by nucleolin (Ncl). Our objective was to identify a novel approach that promotes both DNA synthesis and cytokinesis. Methods: Various techniques, including RNA/protein-sequencing analysis, Ribo-Halo, Ribo-disome, flow cytometry, and cardiac-specific tumor-suppressor retinoblastoma-1 (Rb1) knockout mice, were employed to assess the series signaling of proliferation/cell-cycle reentry and Ribo-bio/cytokinesis. Echocardiography, confocal imaging, and histology were utilized to evaluate cardiac function. Results: Analysis revealed significantly elevated levels of Rb1, bur decreased levels of circASXL1 in the hearts of MI mice compared to control mice. Deletion of Rb1 induces solely cell-cycle reentry, while augmenting the Ribo-bio modulator Ncl leads to cytokinesis. Mechanically, bioinformatics and the loss/gain studies uncovered that circASXL1/CDK6/Rb1 regulates cell-cycle reentry. Moreover, Ribo-Halo, Ribo-disome and circRNA pull-down assays demonstrated that circASXL1 promotes cytokinesis through Ncl/Ribo-bio. Importantly, exosomes derived from umbilical cord mesenchymal stem cells (UMSC-Exo) had the ability to enhance cardiac function by facilitating the coordinated signaling of cell-cycle reentry and Ribo-bio/cytokinesis. These effects were attenuated by silencing circASXL1 in UMSC-Exo. Conclusion: The series signaling of circASXL1/CDK6/Rb1/cell-cycle reentry and circASXL1/Ncl/Ribo-bio/cytokinesis plays a crucial role in cardiac repair. UMSC-Exo effectively repairs infarcted myocardium by stimulating CM cell-cycle reentry and cytokinesis in a circASXL1-dependent manner. This study provides innovative therapeutic strategies targeting the circASXL1 signaling network for MI and offering potential avenues for enhanced cardiac repair.

Nanoscale intracellular ultrastructures affected by osmotic pressure using small-angle X-ray scattering.

Although intracellular ultrastructures have typically been studied using microscopic techniques, it is difficult to observe ultrastructures at the submicron scale of living cells due to spatial resolution (fluorescence microscopy) or high vacuum environment (electron microscopy). We investigate the nanometer scale intracellular ultrastructures of living CHO cells in various osmolality using small-angle X-ray scattering (SAXS), and especially the structures of ribosomes, DNA double helix, and plasma membranes in-cell environment are observed. Ribosomes expand and contract in response to osmotic pressure, and the inter-ribosomal correlation occurs under isotonic and hyperosmolality. The DNA double helix is not dependent on the osmotic pressure. Under high osmotic pressure, the plasma membrane folds into form a multilamellar structure with a periodic length of about 6 nm. We also study the ultrastructural changes caused by formaldehyde fixation, freezing and heating.

Enrichment of rare codons at 5' ends of genes is a spandrel caused by evolutionary sequence turnover and does not improve translation.

Previously, Tuller et al. found that the first 30-50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons. They argued that this slowed translation, and was adaptive because it queued ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined this 5' slow translation 'ramp.' We confirm that 5' regions are slightly enriched for rare codons; in addition, they are depleted for downstream Start codons (which are fast), with both effects contributing to slow 5' translation. However, we also find that the 5' (and 3') ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turnover relatively rapidly. When a new 5' end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5' ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5' end are expressed relatively poorly, and that substituting faster synonymous codons improves expression. Direct experiment shows that slow codons do not prevent downstream ribosome collisions. Further informatic studies suggest that for natural genes, slow 5' ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus, we conclude that slow 5' translation is a 'spandrel'--a non-adaptive consequence of something else, in this case, the turnover of 5' ends in evolution, and it does not improve translation.

A computational workflow to determine drug candidates alternative to aminoglycosides targeting the decoding center of E. coli ribosome.

The global antibiotic resistance problem necessitates fast and effective approaches to finding novel inhibitors to treat bacterial infections. In this study, we propose a computational workflow to identify plausible high-affinity compounds from FDA-approved, investigational, and experimental libraries for the decoding center on the small subunit 30S of the E. coli ribosome. The workflow basically consists of two molecular docking calculations on the intact 30S, followed by molecular dynamics (MD) simulations coupled with MM-GBSA calculations on a truncated ribosome structure. The parameters used in the molecular docking suits, Glide and AutoDock Vina, as well as in the MD simulations with Desmond were carefully adjusted to obtain expected interactions for the ligand-rRNA complexes. A filtering procedure was followed, considering a fingerprint based on aminoglycoside's binding site on the 30S to obtain seven hit compounds either with different clinical usages or aminoglycoside derivatives under investigation, suggested for in vitro studies. The detailed workflow developed in this study promises an effective and fast approach for the estimation of binding free energies of large protein-RNA and ligand complexes.

Keeping it fresh: ribosomal protein SA sustains sarcomeric function via localized translation.

Mechanical stress from cardiomyocyte contraction causes misfolded sarcomeric protein replacement. Sarcomeric maintenance utilizes localized pools of mRNAs and translation machinery, yet the importance of localized translation remains unclear. In this issue of the JCI, Haddad et al. identify the Z-line as a critical site for localized translation of sarcomeric proteins, mediated by ribosomal protein SA (RPSA). RPSA localized ribosomes at Z-lines and was trafficked via microtubules. Cardiomyocyte-specific loss of RPSA in mice resulted in mislocalized protein translation and caused structural dilation from myocyte atrophy. These findings demonstrate the necessity of RPSA-dependent spatially localized translation for sarcomere maintenance and cardiac structure and function.

Thermodynamic profiles for cotranslational trigger factor substrate recognition.

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

Ribosome Pausing Negatively Regulates Protein Translation in Maize Seedlings during Dark-to-Light Transitions.

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following changes in growth conditions, but ribosome-pausing events are less well understood. In this study, we performed ribosome profiling (Ribo-seq) of etiolated maize (Zea mays) seedlings exposed to light for different durations, revealing hundreds of genes specifically regulated at the translation level during the early period of light exposure. We identified over 400 ribosome-pausing events in the dark that were rapidly released after illumination. These results suggested that ribosome pausing negatively regulates translation from specific genes, a conclusion that was supported by a non-targeted proteomics analysis. Importantly, we identified a conserved nucleotide motif downstream of the pausing sites. Our results elucidate the role of ribosome pausing in the control of gene expression in plants; the identification of the cis-element at the pausing sites provides insight into the mechanisms behind translation regulation and potential targets for artificial control of plant translation.

Functional Activity of Isoform 2 of Human eRF1.

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.