Ribosome Dynamics Research Articles

Computational Strategies to Enhance Cell-Free Protein Synthesis Efficiency

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for protein production, with applications ranging from basic research to biotechnology and pharmaceutical development. However, enhancing the efficiency of CFPS systems remains a crucial challenge for realizing their full potential. Computational strategies offer promising avenues for optimizing CFPS efficiency by providing insights into complex biological processes and enabling rational design approaches. This review provides a comprehensive overview of the computational approaches aimed at enhancing CFPS efficiency. The introduction outlines the significance of CFPS and the role of computational methods in addressing efficiency limitations. It discusses mathematical modeling and simulation-based approaches for predicting protein synthesis kinetics and optimizing CFPS reactions. The review also delves into the design of DNA templates, including codon optimization strategies and mRNA secondary structure prediction tools, to improve protein synthesis efficiency. Furthermore, it explores computational techniques for engineering cell-free transcription and translation machinery, such as the rational design of expression systems and the predictive modeling of ribosome dynamics. The predictive modeling of metabolic pathways and the energy utilization in CFPS systems is also discussed, highlighting metabolic flux analysis and resource allocation strategies. Machine learning and artificial intelligence approaches are being increasingly employed for CFPS optimization, including neural network models, deep learning algorithms, and reinforcement learning for adaptive control. This review presents case studies showcasing successful CFPS optimization using computational methods and discusses applications in synthetic biology, biotechnology, and pharmaceuticals. The challenges and limitations of current computational approaches are addressed, along with future perspectives and emerging trends, such as the integration of multi-omics data and advances in high-throughput screening. The conclusion summarizes key findings, discusses implications for future research directions and applications, and emphasizes opportunities for interdisciplinary collaboration. This review offers valuable insights and prospects regarding computational strategies to enhance CFPS efficiency. It serves as a comprehensive resource, consolidating current knowledge in the field and guiding further advancements.

Transcriptomics and weighted protein network analyses of the LRRK2 protein interactome reveal distinct molecular signatures for sporadic and LRRK2 Parkinson’s Disease

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson’s Disease (LRRK2-PD) and an important risk factor for sporadic PD (sPD). Multiple clinical trials are ongoing to evaluate the benefits associated with the therapeutical reduction of LRRK2 kinase activity. In this study, we described the changes of transcriptomic profiles (whole blood mRNA levels) of LRRK2 protein interactors in sPD and LRRK2-PD cases as compared to healthy controls with the aim of comparing the two PD conditions. We went on to model the protein-protein interaction (PPI) network centred on LRRK2, which was weighted to reflect the transcriptomic changes on expression and co-expression levels of LRRK2 protein interactors. Our results showed that LRRK2 interactors present both similar and distinct alterations in expression levels and co-expression behaviours in the sPD and LRRK2-PD cases; suggesting that, albeit being classified as the same disease based on clinical features, LRRK2-PD and sPD display significant differences from a molecular perspective. Interestingly, the similar changes across the two PD conditions result in decreased connectivity within a topological cluster of the LRRK2 PPI network associated with protein metabolism/biosynthesis and ribosomal metabolism suggesting protein homoeostasis and ribosomal dynamics might be affected in both sporadic and familial PD in comparison with controls.

Ribosomal Protein Dynamics and Its Association with Actin Filaments and Local Translation in Axonal Growth Cones of Dorsal Root Ganglia Neurons.

Local translation in growth cones plays a critical role in responses to extracellular stimuli, such as axon guidance cues. We previously showed that brain-derived neurotrophic factor activates translation and enhances novel protein synthesis through the activation of mammalian target of rapamycin complex 1 signaling in growth cones of dorsal root ganglion neurons. In this study, we focused on 40S ribosomal protein S6 (RPS6), 60S ribosomal protein P0/1/2 (RPP0/1/2), and actin filaments to determine how localization of ribosomal proteins changes with overall protein synthesis induced by neurotrophins. Our quantitative analysis using immunocytochemistry and super-resolution microscopy indicated that RPS6, RPP0/1/2, and actin tend to colocalize in the absence of stimulation, and that these ribosomal proteins tend to dissociate from actin and associate with each other when local protein synthesis is enhanced. We propose that this is because stimulation causes ribosomal subunits to associate with each other to form actively translating ribosomes (polysomes). This study further clarifies the role of cytoskeletal components in local translation in growth cones.

Ultrastructural insights into cellular organization, energy storage and ribosomal dynamics of an ammonia-oxidizing archaeon from oligotrophic oceans.

Nitrososphaeria, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their importance in Earth's ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant unanswered question of how the machinery of these organisms underpins metabolic functions. In this study, we combined spherical-chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaeria. Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules: one type is possibly composed of polyphosphate and the other polyhydroxyalkanoate. With precise measurements using cryo-ET, we observed low quantity and density of ribosomes in SCM1 cells, which are in alignment with the documented slow growth of AOA in laboratory cultures. Collectively, these findings provide visual evidence supporting the resilience of AOA in the vast oligotrophic marine environment.

Abstract 7078: Targeting eIF6 to modulate ribosomal subunit association dynamics in cancers

Abstract Deregulation of ribosomal subunit levels and accessibility are hallmarks of cancer. Eukaryotic translation initiation factor 6 (eIF6) is a key regulator of ribosomal subunit association that is over expressed in many cancers including lung, colon, ovarian and breast cancers, and deregulation of eIF6 is correlated with a poor cancer prognosis. Association of eIF6 with 60S is critical for 60S assembly. However, eIF6 must be released from 60S prior to translation to permit inter subunit interactions between 60S and 40S and to facilitate the formation of translationally proficient 80S monosome. Release of eIF6 is deregulated in the ribosomopathy- Shwachman Diamond Syndrome that is predisposed to leukemias. Also, loss of eIF6 markedly delays tumorigenesis without affecting normal growth, which presents eIF6 as a viable therapeutic target. A potential therapeutic strategy is to target the eIF6 and 60S ribosomal interaction interface. Through biophysical analyses we had identified residues that are critical for the interaction between eIF6 and 60S ribosomal subunit. We show that targeting these key residues in the eIF6-60S interaction interface markedly delays colonic cancer growth and inhibits protein synthesis. Furthermore, we show that the levels of other eIFs and the release factors: SBDS, and EFL1-GTPase are unaltered in the mutant cells suggesting that the translational inhibition is primarily attributed to the deregulation of eIF6. Interestingly, targeting eIF6 leads to an upregulation of p53 independent of the DNA damage response, suggesting that ribosomal stress contributes to the stabilization of p53. Previously, it was shown that translation of Cdc42 was upregulated by eIF6 to promote invasiveness. However, Cdc42 levels were unaltered in the mutant cells. Future studies will determine the mRNA substrates that are translationally deregulated in the mutants. We will also determine the effect of disrupting eIF6 function in inhibiting tumor growth and progression in vivo. Category: MCB, Subcategory: MCB07-Gene regulation sub classification: Translational Control Citation Format: Kavya Meena Harish, Poonam Roshan, Aparna Biswas, Stella Anagnos, Riley Luebbers, Sofia Origanti. Targeting eIF6 to modulate ribosomal subunit association dynamics in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7078.

Investigating the correlated dynamics of ribosomes and chromosomes in Escherichia coli

Investigating the correlated dynamics of ribosomes and chromosomes in Escherichia coli

Life after transcription: control of the fate of neurons

The neocortex is a complex structure responsible for higher-order cognitive abilities in mammals. It consists of six cell layers, each comprised of various subtypes of excitatory and inhibitory neurons. These neurons project their axons to specific targets within each layer. All neocortical projection neurons are generated by neural progenitor cells located in the proliferative ventricular zone. In recent decades, significant strides have been made in comprehending the transcriptional programs governing neuronal cell fate specification. Nonetheless, posttranscriptional mechanisms involved in this process remain largely unexplored. Recently, we found that TrkC-T1, an isoform of the TrkC receptor lacking the kinase domain, determines the fate of corticofugal projection neurons (CFuPN). Our study reveals that the balance between TrkC-T1 and TrkC-TK+, a more widely recognized isoform containing the kinase domain, depends on the type of cell within the developing cortex. Additionally, we demonstrate that two RNA-binding proteins, Srsf1 and Elavl1, work in opposition to establish this balance. Additionally, our data suggests that Srsf1 stimulates the CFuPN fate while Elavl1 stimulates the callosal projection neuron (CPN) fate in vivo by regulating the different TrkC-T1 to TrkC-TK+ ratios. In this study, we identified a protein translation-dependent mechanism that governs the cell fate switch in the developing neocortex. Our results demonstrate that Ire1α, the Inositol-Requiring Enzyme 1α, regulates global translation rates in the developing neocortex through its dynamic interaction with the ribosome, as well as the regulation of expression of translation elongation factors eIF4A1 and eEF-2. Inactivation of Ire1α leads to decreased protein synthesis rates that are associated with stalled ribosomes and a reduced number of sites for translation initiation. We demonstrate the distinctive sensitivity of neurons determined for the upper layer to translation rates. While eEF-2 is necessary for cortical lamination, eIF4A1 regulates the attainment of upper layer fate in a mechanism of translational control that is dependent on structural elements embedded in the 5’UTR of genes responsible for determining fate downstream of Ire1α. Our findings reveal the developmental control of ribosome dynamics as post-transcriptional mechanisms that coordinate the establishment of neuronal diversity and the assembly of cortical layers.

ROS-induced ribosome impairment underlies ZAKα-mediated metabolic decline in obesity and aging.

The ribotoxic stress response (RSR) is a signaling pathway in which the p38- and c-Jun N-terminal kinase (JNK)-activating mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα senses stalling and/or collision of ribosomes. Here, we show that reactive oxygen species (ROS)-generating agents trigger ribosomal impairment and ZAKα activation. Conversely, zebrafish larvae deficient for ZAKα are protected from ROS-induced pathology. Livers of mice fed a ROS-generating diet exhibit ZAKα-activating changes in ribosomal elongation dynamics. Highlighting a role for the RSR in metabolic regulation, ZAK-knockout mice are protected from developing high-fat high-sugar (HFHS) diet-induced blood glucose intolerance and liver steatosis. Finally, ZAK ablation slows animals from developing the hallmarks of metabolic aging. Our work highlights ROS-induced ribosomal impairment as a physiological activation signal for ZAKα that underlies metabolic adaptation in obesity and aging.

Bet-hedging: Bacterial ribosome dynamics during growth transitions

It is known that bacteria reduce their ribosome numbers during nutrient starvation. New research shows that this regulation leads to the formation of two subpopulations with distinct ribosomal RNA levels. The distinct levels affect the growth recovery when nutrients become available, suggesting a possible bet-hedging strategy.

Partial spontaneous intersubunit rotations in pretranslocation ribosomes

The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit rotates counterclockwise relative to the large subunit, placing the ribosome into the rotated conformation. Simultaneously, tRNAs move into the hybrid conformation, and the L1 stalk moves inward toward the P-site tRNA. The conformational dynamics of pretranslocation ribosomes were extensively studied by ensemble and single-molecule methods. Different experimental modalities tracking ribosomal subunits, tRNAs, and the L1 stalk showed that pretranslocation ribosomes undergo spontaneous conformational transitions. Thus, peptidyl transfer unlocks the ribosome and decreases an energy barrier for the reverse ribosome rotation during translocation. However, the tracking of translation with ribosomes labeled at rRNA helices h44 and H101 showed a lack of spontaneous rotations in pretranslocation complexes. Therefore, reverse intersubunit rotations occur during EF-G catalyzed translocation. To reconcile these views, we used high-speed single-molecule microscopy to follow translation in real time. We showed spontaneous rotations in puromycin-released h44-H101 dye-labeled ribosomes. During elongation, the h44-H101 ribosomes undergo partial spontaneous rotations. Spontaneous rotations in h44-H101-labeled ribosomes are restricted prior to aminoacyl-tRNA binding. The pretranslocation h44-H101 ribosomes spontaneously exchanged between three different rotational states. This demonstrates that peptidyl transfer unlocks spontaneous rotations and pretranslocation ribosomes can adopt several thermally accessible conformations, thus supporting the Brownian model of translocation.

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.

Decoding and Recoding of mRNA Sequences by the Ribosome.

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.

Translation machinery captured in motion.

Translation accuracy is one of the most critical factors for protein synthesis. It is regulated by the ribosome and its dynamic behavior, along with translation factors that direct ribosome rearrangements to make translation a uniform process. Earlier structural studies of the ribosome complex with arrested translation factors laid the foundation for an understanding of ribosome dynamics and the translation process as such. Recent technological advances in time-resolved and ensemble cryo-EM have made it possible to study translation in real time at high resolution. These methods provided a detailed view of translation in bacteria for all three phases: initiation, elongation, and termination. In this review, we focus on translation factors (in some cases GTP activation) and their ability to monitor and respond to ribosome organization to enable efficient and accurate translation. This article is categorized under: Translation > Ribosome Structure/Function Translation > Mechanisms.

Cryo-EM structure of the fully assembled Elongator complex.

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.

Nascent peptide-induced translation discontinuation in eukaryotes impacts biased amino acid usage in proteomes

Robust translation elongation of any given amino acid sequence is required to shape proteomes. Nevertheless, nascent peptides occasionally destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains can stochastically induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, using budding yeast and a human factor-based reconstituted translation system, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in their N-terminal regions alter canonical ribosome dynamics, stochastically aborting translation. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we find many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk of failure. Indeed, a bioinformatics analysis reveals that the N-terminal regions of ORFs lack D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

Ribosome impairment regulates intestinal stem cell identity via ZAKɑ activation

The small intestine is a rapidly proliferating organ that is maintained by a small population of Lgr5-expressing intestinal stem cells (ISCs). However, several Lgr5-negative ISC populations have been identified, and this remarkable plasticity allows the intestine to rapidly respond to both the local environment and to damage. However, the mediators of such plasticity are still largely unknown. Using intestinal organoids and mouse models, we show that upon ribosome impairment (driven by Rptor deletion, amino acid starvation, or low dose cyclohexamide treatment) ISCs gain an Lgr5-negative, fetal-like identity. This is accompanied by a rewiring of metabolism. Our findings suggest that the ribosome can act as a sensor of nutrient availability, allowing ISCs to respond to the local nutrient environment. Mechanistically, we show that this phenotype requires the activation of ZAKɑ, which in turn activates YAP, via SRC. Together, our data reveals a central role for ribosome dynamics in intestinal stem cells, and identify the activation of ZAKɑ as a critical mediator of stem cell identity.

Dynamics and growth rate implications of ribosomes and mRNAs interaction in E. coli

Understanding how cells grow and adapt under various nutrient conditions is pivotal in the study of biological stoichiometry. Recent studies provide empirical evidence that cells use multiple strategies to maintain an optimal protein production rate under different nutrient conditions. Mathematical models can provide a solid theoretical foundation that can explain experimental observations and generate testable hypotheses to further our understanding of the growth process. In this study, we generalize a modeling framework that centers on the translation process and study its asymptotic behaviors to validate algebraic manipulations involving the steady states. Using experimental results on the growth of E. coli under C-, N-, and P-limited environments, we simulate the expected quantitative measurements to show the feasibility of using the model to explain empirical evidence. Our results support the findings that cells employ multiple strategies to maintain a similar protein production rate across different nutrient limitations. Moreover, we find that the previous study underestimates the significance of certain biological rates, such as the binding rate of ribosomes to mRNA and the transition rate between different ribosomal stages. Furthermore, our simulation shows that the strategies used by cells under C- and P-limitations result in a faster overall growth dynamics than under N-limitation. In conclusion, the general modeling framework provides a valuable platform to study cell growth under different nutrient supply conditions, which also allows straightforward extensions to the coupling of transcription, translation, and energetics to deepen our understanding of the growth process.

Exportin 1 modulates life span by regulating nucleolar dynamics via the autophagy protein LGG-1/GABARAP.

Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.

Ribosomal RNA 2′-O-methylations regulate translation by impacting ribosome dynamics

SignificanceThe presence of RNA chemical modifications has long been known, but their precise molecular consequences remain unknown. 2'-O-methylation is an abundant modification that exists in RNA in all domains of life. Ribosomal RNA (rRNA) represents a functionally important RNA that is heavily modified by 2'-O-methylations. Although abundant at functionally important regions of the rRNA, the contribution of 2'-O-methylations to ribosome activities is unknown. By establishing a method to disturb rRNA 2'-O-methylation patterns, we show that rRNA 2'-O-methylations affect the function and fidelity of the ribosome and change the balance between different ribosome conformational states. Our work links 2'-O-methylation to ribosome dynamics and defines a set of critical rRNA 2'-O-methylations required for ribosome biogenesis and others that are dispensable.

The Structural Dynamics of Translation.

Accurate protein synthesis (translation) relies on translation factors that rectify ribosome fluctuations into a unidirectional process. Understanding this process requires structural characterization of the ribosome and translation-factor dynamics. In the 2000s, crystallographic studies determined high-resolution structures of ribosomes stalled with translation factors, providing a starting point for visualizing translation. Recent progress in single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution of numerous structures sampled in heterogeneous complexes (ensembles). Ensemble and time-resolved cryo-EM have now revealed unprecedented views of ribosome transitions in the three principal stages of translation: initiation, elongation, and termination. This review focuses on how translation factors help achieve high accuracy and efficiency of translation by monitoring distinct ribosome conformations and by differentially shifting the equilibria of ribosome rearrangements for cognate and near-cognate substrates.