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Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates.

ABSTRACT: Variation in translation-elongation kinetics along a transcript's coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, the presence of proline in the ribosome's P- or A-site slows down translation, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized. Here, we study Saccharomyces cerevisiae using a combination of bioinformatics, mutational experiments, and evolutionary analyses, and show that many different pairs of amino acids and their associated tRNA molecules predictably and causally encode translation rate information when these pairs are present in the A- and P-sites of the ribosome independent of other factors known to influence translation speed including mRNA structure, wobble base pairing, tripeptide motifs, positively charged upstream nascent chain residues, and cognate tRNA concentration. The fast-translating pairs of amino acids that we identify are enriched four-fold relative to the slow-translating pairs across Saccharomyces cerevisiae's proteome, while the slow-translating pairs are enriched downstream of domain boundaries. Thus, the chemical identity of amino acid pairs contributes to variability in translation rates, elongation kinetics are causally encoded in the primary structure of proteins, and signatures of evolutionary selection indicate their potential role in co-translational processes.

SUBMITTER: Ahmed N

PROVIDER: S-EPMC8270670 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates

Project description:Variation in translation-elongation kinetics along a transcript’s coding sequence plays an important role in the maintenance of cellular protein homeostasis by regulating co-translational protein folding, localization, and maturation. Translation-elongation speed is influenced by molecular factors within mRNA and protein sequences. For example, when proline is present in the ribosome’s P- or A-site translation slows down, but the effect of other pairs of amino acids, in the context of all 400 possible pairs, has not been characterized. Here, we study Saccharomyces cerevisiae using a combination of mutational experiments, bioinformatics, and evolutionary analyses, and show that many different pairs of amino acids and their associated tRNA molecules predictably and causally encode translation rate information when these pairs are present in the A- and P-sites of the ribosome independent of other factors known to influence translation speed, including mRNA structure, wobble base pairing, tripeptide motifs, positively charged upstream nascent chain residues, and cognate tRNA concentration. The fast-translating pairs of amino acids that we identify are enriched seven-fold relative to the slow-translating pairs across Saccharomyces cerevisiae’s proteome, while the slow-translating pairs are enriched downstream of domain boundaries. Thus, the chemical identity of amino acid pairs contributes to variability in translation rates, elongation kinetics are causally encoded in the primary structure of proteins, and signatures of evolutionary selection indicate their potential role in co-translational processes.

2020-04-20 | GSE133370 | GEO

Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates

Project description:Pairs of amino acids at the P- and A-sites of the ribosome predictably and causally modulate translation-elongation rates

| PRJNA551280 | ENA

Silent substitutions predictably alter translation elongation rates and protein folding efficiencies.

Project description:Genetic code redundancy allows most amino acids to be encoded by multiple codons that are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold toward the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living Escherichia coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, which occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding.

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Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates.

Project description:Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a 1,000 ORFs in exponentially growing wild-type yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and transfer RNA (tRNA) adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein (RP) genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared with other transcripts with equally high ribosome densities.

| S-EPMC6660795 | biostudies-literature

Protein synthesis rates and ribosome occupancies reveal determinants of translation elongation rates

Project description:Although protein synthesis dynamics has been studied both with theoretical models and by profiling ribosome footprints, the determinants of ribosome flux along open reading frames (ORFs) are not fully understood. Combining measurements of protein synthesis rate with ribosome footprinting data, we here inferred translation initiation and elongation rates for over a thousand ORFs in exponentially-growing wildtype yeast cells. We found that the amino acid composition of synthesized proteins is as important a determinant of translation elongation rate as parameters related to codon and tRNA adaptation. We did not find evidence of ribosome collisions curbing the protein output of yeast transcripts, either in high translation conditions associated with exponential growth, or in strains in which deletion of individual ribosomal protein genes leads to globally increased or decreased translation. Slow translation elongation is characteristic of RP-encoding transcripts, which have markedly lower protein output compared to other transcripts with equally high ribosome densities.

2019-09-10 | PXD014357 | Pride

A chemical kinetic basis for measuring translation initiation and elongation rates from ribosome profiling data.

Project description:Analysis methods based on simulations and optimization have been previously developed to estimate relative translation rates from next-generation sequencing data. Translation involves molecules and chemical reactions, hence bioinformatics methods consistent with the laws of chemistry and physics are more likely to produce accurate results. Here, we derive simple equations based on chemical kinetic principles to measure the translation-initiation rate, transcriptome-wide elongation rate, and individual codon translation rates from ribosome profiling experiments. Our methods reproduce the known rates from ribosome profiles generated from detailed simulations of translation. By applying our methods to data from S. cerevisiae and mouse embryonic stem cells, we find that the extracted rates reproduce expected correlations with various molecular properties, and we also find that mouse embryonic stem cells have a global translation speed of 5.2 AA/s, in agreement with previous reports that used other approaches. Our analysis further reveals that a codon can exhibit up to 26-fold variability in its translation rate depending upon its context within a transcript. This broad distribution means that the average translation rate of a codon is not representative of the rate at which most instances of that codon are translated, and it suggests that translational regulation might be used by cells to a greater degree than previously thought.

| S-EPMC6559674 | biostudies-literature

Ribosome inactivation regulates translation elongation in neurons.

Project description:Cellular plasticity is crucial for adapting to ever-changing stimuli. As a result, cells consistently reshape their translatome, and, consequently, their proteome. The control of translational activity has been thoroughly examined at the stage of translation initiation. However, the regulation of ribosome speed in cells is widely unknown. In this study, we utilized a timed ribosome runoff approach, along with proteomics and transmission electron microscopy, to investigate global translation kinetics in cells. We found that ribosome speeds vary among various cell types, such as astrocytes, induced pluripotent human stem cells, human neural stem cells, and human and rat neurons. Of all cell types studied, mature cortical neurons exhibit the highest rate of translation. This finding is particularly remarkable because mature cortical neurons express the eukaryotic elongation factor 2 (eEF2) at lower levels than other cell types. Neurons solve this conundrum by inactivating a fraction of their ribosomes. As a result, the increase in eEF2 levels leads to a reduction of inactive ribosomes and an enhancement of active ones. Processes that alter the demand for active ribosomes, like neuronal excitation, cause increased inactivation of redundant ribosomes in an eEF2-dependent manner. Our data suggest a novel regulatory mechanism in which neurons dynamically inactivate ribosomes to facilitate translational remodeling. These findings have important implications for developmental brain disorders characterized by, among other things, aberrant translation.

| S-EPMC10869266 | biostudies-literature

Three tRNAs on the ribosome slow translation elongation.

Project description:During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor-quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.

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Activation of GCN2 kinase by ribosome stalling links translation elongation with translation initiation.

Project description:Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results demonstrate the critical interplay between translation elongation and initiation in regulating neuron survival during cellular stress.

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Inferring efficiency of translation initiation and elongation from ribosome profiling.

Project description:One of the main goals of ribosome profiling is to quantify the rate of protein synthesis at the level of translation. Here, we develop a method for inferring translation elongation kinetics from ribosome profiling data using recent advances in mathematical modelling of mRNA translation. Our method distinguishes between the elongation rate intrinsic to the ribosome's stepping cycle and the actual elongation rate that takes into account ribosome interference. This distinction allows us to quantify the extent of ribosomal collisions along the transcript and identify individual codons where ribosomal collisions are likely. When examining ribosome profiling in yeast, we observe that translation initiation and elongation are close to their optima and traffic is minimized at the beginning of the transcript to favour ribosome recruitment. However, we find many individual sites of congestion along the mRNAs where the probability of ribosome interference can reach $50\%$. Our work provides new measures of translation initiation and elongation efficiencies, emphasizing the importance of rating these two stages of translation separately.

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