The impact of ribosomal interference, codon usage, and exit tunnel interactions on translation elongation rate variation

ABSTRACTPrevious studies have shown that translation elongation is regulated by multiple factors, but the observed heterogeneity remains only partially explained. To dissect quantitatively the different determinants of elongation speed, we use probabilistic modeling to estimate initiation and local elongation rates from ribosome profiling data. This model-based approach allows us to quantify the extent of interference between ribosomes on the same transcript. We show that neither interference nor the distribution of slow codons is sufficient to explain the observed heterogeneity. Instead, we find that electrostatic interactions between the ribosomal exit tunnel and specific parts of the nascent polypeptide govern the elongation rate variation as the polypeptide makes its initial pass through the tunnel. Once the N-terminus has escaped the tunnel, the hydropathy of the nascent polypeptide within the ribosome plays a major role in modulating the speed. We show that our results are consistent with the biophysical properties of the tunnel.

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Differential bicodon usage in lowly and highly abundant proteins

PeerJ ◽

10.7717/peerj.3081 ◽

2017 ◽

Vol 5 ◽

pp. e3081 ◽

Cited By ~ 12

Author(s):

Luis A. Diambra

Keyword(s):

Codon Usage ◽

Drug Transport ◽

Heterologous Gene Expression ◽

Elongation Rate ◽

Translation Elongation ◽

Synonymous Codons ◽

Domains Of Life ◽

Small Change ◽

Protein Elongation ◽

Codon Pairs

Degeneracy in the genetic code implies that different codons can encode the same amino acid. Usage preference of synonymous codons has been observed in all domains of life. There is much evidence suggesting that this bias has a major role on protein elongation rate, contributing to differential expression and to co-translational folding. In addition to codon usage bias, other preference variations have been observed such as codon pairs. In this paper, I report that codon pairs have significant different frequency usage for coding either lowly or highly abundant proteins. These usage preferences cannot be explained by the frequency usage of the single codons. The statistical analysis of coding sequences of nine organisms reveals that in many cases bicodon preferences are shared between related organisms. Furthermore, it is observed that misfolding in the drug-transport protein, encoded by MDR1 gene, is better explained by a big change in the pause propensity due to the synonymous bicodon variant, rather than by a relatively small change in codon usage. These findings suggest that codon pair usage can be a more powerful framework to understand translation elongation rate, protein folding efficiency, and to improve protocols to optimize heterologous gene expression.

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A New Look at Codon Usage and Protein Expression

10.29007/d4tz ◽

2019 ◽

Author(s):

Gabriel Wright ◽

Anabel Rodriguez ◽

Patricia Clark ◽

Scott Emrich

Keyword(s):

Protein Expression ◽

Codon Usage ◽

Empirical Data ◽

Codon Usage Bias ◽

Elongation Rate ◽

Translation Rate ◽

Translation Elongation ◽

Promising Alternative ◽

Highly Expressed Genes ◽

Adaptation Index

%MinMax, a model of intra-gene translational elongation rate, relies on codon usage frequencies. Historically, %MinMax has used tables that measure codon usage bias for all genes in an organism, such as those found at HIVE-CUT. In this paper, we provide evidence that codon usage bias based on all genes is insufficient to accurately measure absolute translation rate. We show that alternative ”High-φ” codon usage tables, generated by another model (ROC-SEMPPR), are a promising alternative. By creating a hybrid model, future codon usage analyses and their applications (e.g., codon harmonization) are likely to more accurately measure the ”tempo” of translation elongation. We also suggest a High- φ alternative to the Codon Adaptation Index (CAI), a classic metric of codon usage bias based on highly expressed genes. Significantly, our new alternative is equally well correlated with empirical data as traditional CAI without using experimentally determined expression counts as input.

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Ribosome exit tunnel electrostatics

10.1101/2020.10.20.346684 ◽

2020 ◽

Author(s):

Marc Joiret ◽

Francesca Rapino ◽

Pierre Close ◽

Liesbet Geris

Keyword(s):

Translational Control ◽

Elongation Rate ◽

Electrostatic Model ◽

Amino Acid Residues ◽

Physical Description ◽

Axial Forces ◽

Entry Points ◽

Exit Tunnel ◽

Ribosome Exit Tunnel ◽

The Impact

AbstractThe impact of the ribosome exit tunnel electrostatics on the protein elongation rate or on the forces acting upon the nascent polypeptide chain are currently not fully elucidated. In the past, researchers have measured the electrostatic potential inside the ribosome polypeptide exit tunnel at a limited number of spatial points, at least in prokaryotes. Here, we present a basic electrostatic model of the exit tunnel of the ribosome, providing a quantitative physical description of the tunnel interaction with the nascent proteins at all centro-axial points inside the tunnel. We show how the tunnel geometry causes a positive potential difference between the tunnel exit and entry points which impedes positively charged amino acid residues from progressing through the tunnel, affecting the elongation rate in a range of minus 40% to plus 85% when compared to the average elongation rate. The time spent by the ribosome to decode the genetic encrypted message is constrained accordingly. We quantitatively derived, at single residue resolution, the axial forces acting on the nascent peptide from its particular sequence embedded in the tunnel. The model sheds light on how the experimental data point measurements of the potential are linked to the local structural chemistry of the inner wall and the shape and size of the tunnel. The model consistently connects experimental observations coming from different fields in molecular biology, structural and physical chemistry, biomechanics, synthetic and multi-omics biology. Our model should be a valuable tool to gain insight into protein synthesis dynamics, translational control and into the role of the ribosome’s mechanochemistry in the co-translational protein folding.

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Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

Genome Biology ◽

10.1186/s13059-020-02256-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Taolan Zhao ◽

Yan-Ming Chen ◽

Yu Li ◽

Jia Wang ◽

Siyu Chen ◽

...

Keyword(s):

Protein Quality ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Translation Elongation ◽

Cryo Electron Microscopy ◽

Cotranslational Folding ◽

A Cell ◽

Exit Tunnel ◽

Hidden Layer ◽

A Site

Abstract Background The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. Results In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. Conclusions Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

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

Vaccines ◽

10.3390/vaccines9070734 ◽

2021 ◽

Vol 9 (7) ◽

pp. 734

Author(s):

Xuhua Xia

Keyword(s):

Codon Usage ◽

Translation Initiation ◽

Mrna Stability ◽

Stop Codon ◽

Translation Termination ◽

Critical Evaluation ◽

Immunization Programs ◽

Translation Elongation ◽

Different Types ◽

Similarities And Differences

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.

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Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽

10.1186/s13568-019-0890-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Neeraja Punde ◽

Jennifer Kooken ◽

Dagmar Leary ◽

Patricia M. Legler ◽

Evelina Angov

Keyword(s):

Protein Structure ◽

Amino Acid ◽

Codon Usage ◽

Dna Sequences ◽

Structural Integrity ◽

Host Cells ◽

Loss Of Function ◽

Species Specific ◽

And Function ◽

The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

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Intragenomic variation in mutation biases causes underestimation of selection on synonymous codon usage

10.1101/2021.10.29.466462 ◽

2021 ◽

Author(s):

Alexander L Cope ◽

Premal Shah

Keyword(s):

Population Genetics ◽

Natural Selection ◽

Codon Usage ◽

Codon Bias ◽

Synonymous Codon ◽

Synonymous Codon Usage ◽

Mutation Bias ◽

Biased Gene Conversion ◽

Intragenomic Variation ◽

The Impact

Patterns of non-uniform usage of synonymous codons (codon bias) varies across genes in an organism and across species from all domains of life. The bias in codon usage is due to a combination of both non-adaptive (e.g. mutation biases) and adaptive (e.g. natural selection for translation efficiency/accuracy) evolutionary forces. Most population genetics models quantify the effects of mutation bias and selection on shaping codon usage patterns assuming a uniform mutation bias across the genome. However, mutation biases can vary both along and across chromosomes due to processes such as biased gene conversion, potentially obfuscating signals of translational selection. Moreover, estimates of variation in genomic mutation biases are often lacking for non-model organisms. Here, we combine an unsupervised learning method with a population genetics model of synonymous codon bias evolution to assess the impact of intragenomic variation in mutation bias on the strength and direction of natural selection on synonymous codon usage across 49 Saccharomycotina budding yeasts. We find that in the absence of a priori information, unsupervised learning approaches can be used to identify regions evolving under different mutation biases. We find that the impact of intragenomic variation in mutation bias varies widely, even among closely-related species. We show that the overall strength and direction of selection on codon usage can be underestimated by failing to account for intragenomic variation in mutation biases. Interestingly, genes falling into clusters identified by machine learning are also often physically clustered across chromosomes, consistent with processes such as biased gene conversion. Our results indicate the need for more nuanced models of sequence evolution that systematically incorporate the effects of variable mutation biases on codon frequencies.

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Cast Iron Cooling Rate Variation Using Cooling Channels: A Numerical Approach

Journal of Engineering Research and Reports ◽

10.9734/jerr/2021/v20i417292 ◽

2021 ◽

pp. 29-35

Author(s):

Olumide Adewole Towoju

Keyword(s):

Mechanical Properties ◽

Cast Iron ◽

Cooling Rate ◽

Flow Rate ◽

Numerical Approach ◽

Cooling Curve ◽

Rate Variation ◽

Cooling Fluid ◽

Cooling Channels ◽

The Impact

The cooling rate of molten cast iron can make or mar it. The cooling rate plays a significant role in the resulting mechanical properties of cast iron. It determines the grain growth and size. The mechanical properties of cast iron variation along its length are achieved either with the use of different mold materials or by sectioning to ensure varied cooling rates. Mechanical properties can, however, also be varied along its length without any of these adopted methods by the incorporation of cooling channels in the mould. This study seeks to expand the frontier of this concept with the use of different cooling fluids and fluid flow rate, and numerically investigate the impact on the cooling rate of gray cast iron (class 40). The cooling curve for the cast iron was impacted by the use of different cooling fluids with the attainment of the desired mechanical properties with the selection of an appropriate cooling fluid. Also, the flow rate of the cooling fluid has an impact on the cast iron cooling rate.

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