EEF3 promotes late stages of tRNA translocation on the ribosome
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE160206 | GEO | 2020/10/28
REPOSITORIES: GEO
Similar Datasets
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115161 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115160 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115159 | GEO
Project description:Ribosomes undergo substantial conformational changes during translation elongation to accommodate incoming aminoacyl-tRNAs and translocate along the mRNA template. We used multiple elongation inhibitors and chemical probing to define ribosome conformational states corresponding to different sized ribosome-protected mRNA fragments (RPFs) generated by ribosome profiling. We show using various genetic and environmental perturbations that the previously identified 20-22 nucleotide (nt) RPFs correspond predominantly to ribosomes in a pre-accommodation state with an open 40S ribosomal A site while the classical 27-29 nt fragments correspond to ribosomes in a pre-translocation state with an occupied 40S ribosomal A site. These distinct ribosome conformational states revealed by ribosome profiling are seen in all eukaryotes tested including fungi, worms and mammals. This high-resolution ribosome profiling approach reveals the anticipated Rck2-dependent inhibition of translocation through eEF2 phosphorylation during hyperosmotic stress. These same approaches reveal a strong translation elongation arrest during oxidative stress where the ribosomes are trapped in a pre-translocation state, but in this case the translational arrest is independent of Rck2-driven eEF2 phosphorylation. These results provide new insights and approaches for defining the molecular events that impact translation elongation throughout biology.
2019-01-27 | GSE115158 | GEO
Project description:Firczuk2013 - Eukaryotic mRNA translation machinery
This is a model of Saccharomyces cerevisiae
mRNA translation which includes the initiation, elongation and termination phases. The model is for 20 condon mRNAs. The building of a multi-factor complex in initiation and also the different processes in elongation and termination are modelled in detail. The model takes into account that ribosomes cover more than one codon of mRNA so that the movement of ribosomes are effectively blocked by other ribosomes several codons downstream. It is assumed that 15 codons are occupied by each ribosome. This blocking effect is considered in reaction R18 in initiation and also reaction R26, the reaction where translocation of ribosomes takes place in elongation. The kinetic functions of these two reactions are based on MacDonald et al. 1968 and Heinrich & Rapaport 1980. All other kinetic functions follow mass-action kinetics. The concentrations of transfer RNA species (Met-tRNA, aa-tRNA and tRNA in the model) are kept constant, while the other species' concentrations can change in the course of the simulation. The model describes the translation of a short mRNA with 20 codons. Therefore, all reactions in the elongation cycle (R22, R23, R25, R26, R28 and R29) and the corresponding species are replicated accordingly to model the species with ribosomes bound at different positions. In summary, the model contains 165 different species and 141 reactions.
The value of the 56 rate constant parameters were estimated by fitting the model against a series of experimental data consisting of modulation of the various translation factors (Figures 2, 3 and S3). Overall the parameter estimation was carried out over 212 different data points (steady states).
This model is described in the article:
An in vivo control map for the eukaryotic mRNA translation machinery
Helena Firczuk, Shichina Kannambath, Jürgen Pahle, Amy Claydon, Robert Beynon, John Duncan, Hans Westerhoff, Pedro Mendes and John EG McCarthy
Molecular Systems Biology. 9:635
Abstract:
Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
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and identified by: BIOMD0000000457
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2011-04-30 | BIOMD0000000457 | BioModels
Project description:Eukaryotic elongation factor 1 alpha (eEF1A) delivers aminoacyl-tRNA to the ribosome and thereby plays a key role in protein synthesis. Human eEF1A is post-translationally methylated on several lysine residues as well as on the N-terminus and until recently the responsible methyltransferases were largely elusive. Using mass spectrometry based screens, gene targeted cells and in vitro enzymology we identify the human methyltransferase like proteins 13 (METTL13), which contains two distinct methyltransferase domains, as an enzyme catalyzing both dimethylation of Lys55 and trimethylation of the N-terminus of eEF1A. Moreover, through ribosome profiling we demonstrate that loss of METTL13 function alters translation dynamics and results in changed translation rate of specific codons. In line with the established descriptive nomenclature for this type of enzymes we suggest that METTL13 is redubbed eEF1A dual methyltransferase (eEF1A-DMT / EEF1ADMT).
2018-07-04 | GSE104033 | GEO
Project description:Diphthamide (DPH) is a conserved amino acid modification on eukaryotic translation elongation factor eEF2. We show that loss of DPH increases -1 ribosomal frameshifting at both programmed, including in SARS-CoV-2, and non-programmed sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation. The drop-off defect on the long yeast MDN1 gene was suppressed by eliminating out-of-frame stop codons. Our data reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting and premature termination at out-of-frame stop codons.
2023-01-10 | GSE176169 | GEO
Project description:Chromatin remodeling and protein synthesis are tightly regulated processes that impact gene expression and cellular phenotypes. However, it is unknown if these two regulatory mechanisms are linked or exert independent effects in normal epithelial physiology and disease states. We have uncovered a new functional relationship between the chromatin remodeler ARID1A and mRNA translation elongation that is involved in maintaining cellular fitness in the context of bladder carcinogenesis. Loss of ARID1A triggers inhibition of the translation elongation factor eEF2 and ribosome accumulation, which results in a reduction in protein synthesis. In the setting of ARID1A deficiency the bladder carcinogen BBN induces DNA damage and cell death without impacting cancer initiation. As such, ARID1A is necessary for tumorigenesis. Remarkably, restoration of translation elongation through eEF2 is sufficient to initiate transformation in the context of ARID1A loss. However, dependence on ARID1A for transformation appears to be context specific, as loss of ARID1A after tumor establishment is sufficient for cancer progression.
2023-03-13 | GSE201220 | GEO
Project description:Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many nonhistone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1Amethylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosome profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.
2017-01-05 | GSE93133 | GEO
Project description:Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we find that the universal cotranslational machinery adapts to accommodate the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). During eEF1A synthesis, chaperone Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult to fold protein that necessitates dedicated folding factor Chp1 at the ribosomal tunnel exit to protect the eukaryotic cell from proteostasis collapse.
2024-01-16 | GSE221651 | GEO