RESUMEN
A novel class of protein misfolding characterized by either the formation of non-native noncovalent lasso entanglements in the misfolded structure or loss of native entanglements has been predicted to exist and found circumstantial support through biochemical assays and limited-proteolysis mass spectrometry data. Here, we examine whether it is possible to design small molecule compounds that can bind to specific folding intermediates and thereby avoid these misfolded states in computer simulations under idealized conditions (perfect drug-binding specificity, zero promiscuity, and a smooth energy landscape). Studying two proteins, type III chloramphenicol acetyltransferase (CAT-III) and D-alanyl-D-alanine ligase B (DDLB), that were previously suggested to form soluble misfolded states through a mechanism involving a failure-to-form of native entanglements, we explore two different drug design strategies using coarse-grained structure-based models. The first strategy, in which the native entanglement is stabilized by drug binding, failed to decrease misfolding because it formed an alternative entanglement at a nearby region. The second strategy, in which a small molecule was designed to bind to a non-native tertiary structure and thereby destabilize the native entanglement, succeeded in decreasing misfolding and increasing the native state population. This strategy worked because destabilizing the entanglement loop provided more time for the threading segment to position itself correctly to be wrapped by the loop to form the native entanglement. Further, we computationally identified several FDA-approved drugs with the potential to bind these intermediate states and rescue misfolding in these proteins. This study suggests it is possible for small molecule drugs to prevent protein misfolding of this type.
Asunto(s)
Pliegue de Proteína , Proteínas , Proteínas/química , Simulación por Computador , Programas Informáticos , Espectrometría de MasasRESUMEN
Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.
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Membrana Celular , Escherichia coli , Exorribonucleasas , Multimerización de Proteína , Mutación Silenciosa , Escherichia coli/enzimología , Exorribonucleasas/química , Exorribonucleasas/genética , Cinética , Pliegue de Proteína , Multimerización de Proteína/genética , Membrana Celular/enzimologíaRESUMEN
The binding of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to the host cell receptor angiotensin-converting enzyme 2 (ACE2) is the first step in human viral infection. Therefore, understanding the mechanism of interaction between RBD and ACE2 at the molecular level is critical for the prevention of COVID-19, as more variants of concern, such as Omicron, appear. Recently, atomic force microscopy has been applied to characterize the free energy landscape of the RBD-ACE2 complex, including estimation of the distance between the transition state and the bound state, xu. Here, using a coarse-grained model and replica-exchange umbrella sampling, we studied the free energy landscape of both the wild type and Omicron subvariants BA.1 and XBB.1.5 interacting with ACE2. In agreement with experiment, we find that the wild type and Omicron subvariants have similar xu values, but Omicron binds ACE2 more strongly than the wild type, having a lower dissociation constant KD.
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COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , Enzima Convertidora de Angiotensina 2 , Mutación , Unión ProteicaRESUMEN
One-third of protein domains in the CATH database contain a recently discovered tertiary topological motif: non-covalent lasso entanglements, in which a segment of the protein backbone forms a loop closed by non-covalent interactions between residues and is threaded one or more times by the N- or C-terminal backbone segment. Unknown is how frequently this structural motif appears across the proteomes of organisms. And the correlation of these motifs with various classes of protein function and biological processes have not been quantified. Here, using a combination of protein crystal structures, AlphaFold2 predictions, and Gene Ontology terms we show that in E. coli, S. cerevisiae and H. sapiens that 71%, 52% and 49% of globular proteins contain one-or-more non-covalent lasso entanglements in their native fold, and that some of these are highly complex with multiple threading events. Further, proteins containing these tertiary motifs are consistently enriched in certain functions and biological processes across these organisms and depleted in others, strongly indicating an influence of evolutionary selection pressures acting positively and negatively on the distribution of these motifs. Together, these results demonstrate that non-covalent lasso entanglements are widespread and indicate they may be extensively utilized for protein function and subcellular processes, thus impacting phenotype.
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Bases de Datos de Proteínas , Evolución Molecular , Pliegue de Proteína , Proteoma , Escherichia coli , Proteoma/química , Saccharomyces cerevisiae/genética , Humanos , Dominios ProteicosRESUMEN
BACKGROUND: Synonymous mutations, which change the DNA sequence but not the encoded protein sequence, can affect protein structure and function, mRNA maturation, and mRNA half-lives. The possibility that synonymous mutations might be enriched in cancer has been explored in several recent studies. However, none of these studies control for all three types of mutational heterogeneity (patient, histology, and gene) that are known to affect the accurate identification of non-synonymous cancer-associated genes. Our goal is to adopt the current standard for non-synonymous mutations in an investigation of synonymous mutations. RESULTS: Here, we create an algorithm, MutSigCVsyn, an adaptation of MutSigCV, to identify cancer-associated genes that are enriched for synonymous mutations based on a non-coding background model that takes into account the mutational heterogeneity across these levels. Using MutSigCVsyn, we first analyzed 2572 cancer whole-genome samples from the Pan-cancer Analysis of Whole Genomes (PCAWG) to identify non-synonymous cancer drivers as a quality control. Indicative of the algorithm accuracy we find that 58.6% of these candidate genes were also found in Cancer Census Gene (CGC) list, and 66.2% were found within the PCAWG cancer driver list. We then applied it to identify 30 putative cancer-associated genes that are enriched for synonymous mutations within the same samples. One of the promising gene candidates is the B cell lymphoma 2 (BCL-2) gene. BCL-2 regulates apoptosis by antagonizing the action of proapoptotic BCL-2 family member proteins. The synonymous mutations in BCL2 are enriched in its anti-apoptotic domain and likely play a role in cancer cell proliferation. CONCLUSION: Our study introduces MutSigCVsyn, an algorithm that accounts for mutational heterogeneity at patient, histology, and gene levels, to identify cancer-associated genes that are enriched for synonymous mutations using whole genome sequencing data. We identified 30 putative candidate genes that will benefit from future experimental studies on the role of synonymous mutations in cancer biology.
Asunto(s)
Neoplasias , Mutación Silenciosa , Humanos , Genoma Humano , Mutación , Neoplasias/patología , ARN Mensajero , Proteínas Proto-Oncogénicas c-bcl-2 , Análisis Mutacional de ADNRESUMEN
Subpopulations of soluble, misfolded proteins can bypass chaperones within cells. The extent of this phenomenon and how it happens at the molecular level are unknown. Through a meta-analysis of the experimental literature we find that in all quantitative protein refolding studies there is always a subpopulation of soluble but misfolded protein that does not fold in the presence of one or more chaperones, and can take days or longer to do so. Thus, some misfolded subpopulations commonly bypass chaperones. Using multi-scale simulation models we observe that the misfolded structures that bypass various chaperones can do so because their structures are highly native like, leading to a situation where chaperones do not distinguish between the folded and near-native-misfolded states. More broadly, these results provide a mechanism by which long-time scale changes in protein structure and function can persist in cells because some misfolded states can bypass components of the proteostasis machinery.
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Chaperonas Moleculares , Pliegue de Proteína , Chaperonas Moleculares/metabolismoRESUMEN
The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular machine responsible for protein synthesis, is known to interact with nascent proteins, adding further complexity to the protein folding landscape. Consequently, it is unclear whether the folding pathways of proteins are conserved on and off the ribosome. The main question remains: to what extent does the ribosome help proteins fold? To address this question, we used coarse-grained molecular dynamics simulations to compare the mechanisms by which the proteins dihydrofolate reductase, type III chloramphenicol acetyltransferase, and d-alanine-d-alanine ligase B fold during and after vectorial synthesis on the ribosome to folding from the full-length unfolded state in bulk solution. Our results reveal that the influence of the ribosome on protein folding mechanisms varies depending on the size and complexity of the protein. Specifically, for a small protein with a simple fold, the ribosome facilitates efficient folding by helping the nascent protein avoid misfolded conformations. However, for larger and more complex proteins, the ribosome does not promote folding and may contribute to the formation of intermediate misfolded states cotranslationally. These misfolded states persist posttranslationally and do not convert to the native state during the 6 µs runtime of our coarse-grain simulations. Overall, our study highlights the complex interplay between the ribosome and protein folding and provides insight into the mechanisms of protein folding on and off the ribosome.
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Biosíntesis de Proteínas , Pliegue de Proteína , Ribosomas/metabolismo , Proteínas/metabolismo , Simulación de Dinámica MolecularRESUMEN
The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.
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Biosíntesis de Proteínas , Mutación Silenciosa , Codón/metabolismo , ARN Mensajero/genética , Ribosomas/metabolismo , Escherichia coli/genéticaRESUMEN
Conventional quantum mechanical-molecular mechanics (QM/MM) simulation approaches for modeling enzyme reactions often assume that there is one dominant reaction pathway and that this pathway can be sampled starting from an X-ray structure of the enzyme. These assumptions reduce computational cost; however, their validity has not been extensively tested. This is due in part to the lack of a rigorous formalism for integrating disparate pathway information from dynamical QM/MM calculations. Here, we present a way to model ensembles of reaction pathways efficiently using a divide-and-conquer strategy through Hierarchical Markov State Modeling (Hi-MSM). This approach allows information on multiple, distinct pathways to be incorporated into a chemical kinetic model, and it allows us to test these two assumptions. Applying Hi-MSM to the reaction carried out by dihydrofolate reductase (DHFR) we find (i) there are multiple, distinct pathways significantly contributing to the overall flux of the reaction that the conventional approach does not identify and (ii) that the conventional approach does not identify the dominant reaction pathway. Thus, both assumptions underpinning the conventional approach are violated. Since DHFR is a relatively small enzyme, and configuration space scales exponentially with protein size, accounting for multiple reaction pathways is likely to be necessary for most enzymes.
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Modelos Químicos , Tetrahidrofolato Deshidrogenasa , Cinética , Tetrahidrofolato Deshidrogenasa/química , Química Física , Simulación de Dinámica Molecular , Teoría CuánticaRESUMEN
Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured. These proteins are abundant and highly structured. Notably, they display a tendency to form ß sheet secondary structures, have more complex folding topology, and are enriched for chaperone-binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides are also more often components of stable protein complexes in comparison with other proteins. Combining these findings suggests the existence of a specific subset of proteins in the cell that is particularly vulnerable to misfolding and aggregation following synthesis before reaching the native state.
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Pliegue de Proteína , Proteoma , Chaperonas Moleculares/metabolismo , Péptidos/metabolismo , Unión Proteica , Proteoma/metabolismoRESUMEN
Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.
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Proteínas de Escherichia coli , Proteostasis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , ProteolisisRESUMEN
A promising approach to combat Covid-19 infections is the development of effective antiviral antibodies that target the SARS-CoV-2 spike protein. Understanding the structures and molecular mechanisms underlying the binding of antibodies to SARS-CoV-2 can contribute to quickly achieving this goal. Recently, a cocktail of REGN10987 and REGN10933 antibodies was shown to be an excellent candidate for the treatment of Covid-19. Here, using all-atom steered molecular dynamics and coarse-grained umbrella sampling, we examine the interactions of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with REGN10987 and REGN10933 separately as well as together. Both computational methods show that REGN10933 binds to RBD more strongly than REGN10987. Importantly, the cocktail binds to RBD (simultaneous binding) more strongly than its components. The dissociation constants of REGN10987-RBD and REGN10933-RBD complexes calculated from the coarse-grained simulations are in good agreement with the experimental data. Thus, REGN10933 is probably a better candidate for treating Covid-19 than REGN10987, although the cocktail appears to neutralize the virus more efficiently than REGN10933 or REGN10987 alone. The association of REGN10987 with RBD is driven by van der Waals interactions, while electrostatic interactions dominate in the case of REGN10933 and the cocktail. We also studied the effectiveness of these antibodies on the two most dangerous variants Delta and Omicron. Consistent with recent experimental reports, our results confirmed that the Omicron variant reduces the neutralizing activity of REGN10933, REGN10987, and REGN10933+REGN10987 with the K417N, N440K, L484A, and Q498R mutations playing a decisive role, while the Delta variant slightly changes their activity.
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COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Humanos , Glicoproteína de la Espiga del CoronavirusRESUMEN
A genetic knockout can be lethal to one human cell type while increasing growth rate in another. This context specificity confounds genetic analysis and prevents reproducible genome engineering. Genome-wide CRISPR compendia across most common human cell lines offer the largest opportunity to understand the biology of cell specificity. The prevailing viewpoint, synthetic lethality, occurs when a genetic alteration creates a unique CRISPR dependency. Here, we use machine learning for an unbiased investigation of cell type specificity. Quantifying model accuracy, we find that most cell type specific phenotypes are predicted by the function of related genes of wild-type sequence, not synthetic lethal relationships. These models then identify unexpected sets of 100-300 genes where reduced CRISPR measurements can produce genome-scale loss-of-function predictions across >18,000 genes. Thus, it is possible to reduce in vitro CRISPR libraries by orders of magnitude-with some information loss-when we remove redundant genes and not redundant sgRNAs.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ingeniería Genética , Aprendizaje Automático , Animales , Línea Celular , Técnicas de Inactivación de Genes , Genómica , Humanos , Mutaciones Letales SintéticasRESUMEN
The presence of a single cluster of nonoptimal codons was found to decrease a transcript's half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae The impact of multiple nonoptimal codon clusters on a transcript's half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5' end can lead to synergistic effects that increase a messenger RNA's (mRNA's) half-life in S. cerevisiae Specifically, the 5' end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5' end is the primary determinant of mRNA half-life. These results suggest the "translational ramp," in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.
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Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/biosíntesis , Semivida , Modelos GenéticosRESUMEN
The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multiscale simulations, that positively charged residues generate large forces that move the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but move the A- and P-sites closer together. These conformational changes, respectively, increase and decrease the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation and provide a framework for interpreting experimental results on translation speed.
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Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Ribosomas/fisiología , Aminoácidos/metabolismo , Cinética , Modelos Químicos , Modelos Teóricos , Conformación Proteica , Ribosomas/metabolismo , Ribosomas/ultraestructura , Electricidad EstáticaRESUMEN
Interactions between the ribosome and nascent chain can destabilize folded domains in the ribosome exit tunnel's vestibule, the last 3 nm of the exit tunnel where tertiary folding can occur. Here, we test if a contribution to this destabilization is a weakening of hydrophobic association, the driving force for protein folding. Using all-atom molecular dynamics simulations, we calculate the potential-of-mean force between two methane molecules along the center line of the ribosome exit tunnel and in bulk solution. Associated methanes, we find, are half as stable in the ribosome's vestibule as compared to bulk solution, demonstrating that the hydrophobic effect is weakened by the presence of the ribosome. This decreased stability arises from a decrease in the amount of water entropy gained upon the association of the methanes. And this decreased entropy gain originates from water molecules being more ordered in the vestibule as compared to bulk solution. Therefore, the hydrophobic effect is weaker in the vestibule because waters released from the first solvation shell of methanes upon association do not gain as much entropy in the vestibule as they do upon release in bulk solution. These findings mean that nascent proteins pass through a ribosome vestibule environment that can destabilize folded structures, which has the potential to influence co-translational protein folding pathways, energetics, and kinetics.
RESUMEN
A structural understanding of the mechanism by which antibodies bind SARS-CoV-2 at the atomic level is highly desirable as it can tell the development of more effective antibodies to treat Covid-19. Here, we use steered molecular dynamics (SMD) and coarse-grained simulations to estimate the binding affinity of the monoclonal antibodies CR3022 and 4A8 to the SARS-CoV-2 receptor-binding domain (RBD) and SARS-CoV-2 N-terminal domain (NTD). Consistent with experiments, our SMD and coarse-grained simulations both indicate that CR3022 has a higher affinity for SARS-CoV-2 RBD than 4A8 for the NTD, and the coarse-grained simulations indicate the former binds three times stronger to its respective epitope. This finding shows that CR3022 is a candidate for Covid-19 therapy and is likely a better choice than 4A8. Energetic decomposition of the interaction energies between these two complexes reveals that electrostatic interactions explain the difference in the observed binding affinity between the two complexes. This result could lead to a new approach for developing anti-Covid-19 antibodies in which good candidates must contain charged amino acids in the area of contact with the virus.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Antivirales , Humanos , SARS-CoV-2 , Electricidad EstáticaRESUMEN
Mechanical forces acting on the nascent chain residue located at the P-site of the ribosome can influence codon translation rates. Most observations to date involve force vectors aligned collinear with the long axis of the ribosome exit tunnel. What is poorly understood is how force applied in other directions will impact the rate of peptide bond formation catalyzed by the ribosome. Here, we utilize quantum mechanical/molecular mechanics simulations to estimate the changes in the activation free energy as a consequence of applying a constant force in various directions on the C-terminal residue at the P-site. Qualitatively consistent with the Bell model, we find this force can either accelerate, decelerate, or not alter the reaction rate depending on the force direction. A force in the average direction between the P-site 3' O-C ester bond that breaks and the peptide bond that forms accelerates the reaction. A force in the opposite direction slows down the reaction as it opposes these bonds breaking and forming, but surprisingly it does not do so to the maximum extent possible. In this case, there is a counterbalancing trend; the force in this direction brings the A-site amino nitrogen and the P-site tRNA A76 3' oxygen groups closer together, which promotes one of the proton shuttling steps of the reaction. We find the maximum force-induced slowdown occurs 37° off this axis. If force is applied in orthogonal directions to the reaction coordinates, there is no significant change in the reaction speed. These results indicate that there is a richer set of scenarios of force effects on translation speed that have yet to be experimentally explored and raise the possibility that cells could use these mechanochemical effects to modulate and regulate protein synthesis.
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ARN de Transferencia , Ribosomas , Catálisis , Fenómenos Mecánicos , Simulación de Dinámica Molecular , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
BACKGROUND: Translation is a fundamental process in gene expression. Ribosome profiling is a method that enables the study of transcriptome-wide translation. A fundamental, technical challenge in analyzing Ribo-Seq data is identifying the A-site location on ribosome-protected mRNA fragments. Identification of the A-site is essential as it is at this location on the ribosome where a codon is translated into an amino acid. Incorrect assignment of a read to the A-site can lead to lower signal-to-noise ratio and loss of correlations necessary to understand the molecular factors influencing translation. Therefore, an easy-to-use and accurate analysis tool is needed to accurately identify the A-site locations. RESULTS: We present RiboA, a web application that identifies the most accurate A-site location on a ribosome-protected mRNA fragment and generates the A-site read density profiles. It uses an Integer Programming method that reflects the biological fact that the A-site of actively translating ribosomes is generally located between the second codon and stop codon of a transcript, and utilizes a wide range of mRNA fragment sizes in and around the coding sequence (CDS). The web application is containerized with Docker, and it can be easily ported across platforms. CONCLUSIONS: The Integer Programming method that RiboA utilizes is the most accurate in identifying the A-site on Ribo-Seq mRNA fragments compared to other methods. RiboA makes it easier for the community to use this method via a user-friendly and portable web application. In addition, RiboA supports reproducible analyses by tracking all the input datasets and parameters, and it provides enhanced visualization to facilitate scientific exploration. RiboA is available as a web service at https://a-site.vmhost.psu.edu/ . The code is publicly available at https://github.com/obrien-lab/aip_web_docker under the MIT license.