Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation.

In bacterial translational initiation, three initiation factors (IFs 1-3) enable the selection of initiator tRNA and the start codon in the P site of the 30S ribosomal subunit. Here, we report 11 single-particle cryo-electron microscopy (cryoEM) reconstructions of the complex of bacterial 30S subunit with initiator tRNA, mRNA, and IFs 1-3, representing different steps along the initiation pathway. IF1 provides key anchoring points for IF2 and IF3, thereby enhancing their activities. IF2 positions a domain in an extended conformation appropriate for capturing the formylmethionyl moiety charged on tRNA. IF3 and tRNA undergo large conformational changes to facilitate the accommodation of the formylmethionyl-tRNA (fMet-tRNA(fMet)) into the P site for start codon recognition.

Structural changes enable start codon recognition by the eukaryotic translation initiation complex.

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes.

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

The initiation factor 3 (IF3) residues interacting with initiator tRNA elbow modulate the fidelity of translation initiation and growth fitness in Escherichia coli.

Initiation factor 3 (IF3) regulates the fidelity of bacterial translation initiation by debarring the use of non-canonical start codons or non-initiator tRNAs and prevents premature docking of the 50S ribosomal subunit to the 30S pre-initiation complex (PIC). The C-terminal domain (CTD) of IF3 can carry out most of the known functions of IF3 and sustain Escherichia coli growth. However, the roles of the N-terminal domain (NTD) have remained unclear. We hypothesized that the interaction between NTD and initiator tRNAfMet (i-tRNA) is essential to coordinate the movement of the two domains during the initiation pathway to ensure fidelity of the process. Here, using atomistic molecular dynamics (MD) simulation, we show that R25A/Q33A/R66A mutations do not impact NTD structure but disrupt its interaction with i-tRNA. These NTD residues modulate the fidelity of translation initiation and are crucial for bacterial growth. Our observations also implicate the role of these interactions in the subunit dissociation activity of CTD of IF3. Overall, the study shows that the interactions between NTD of IF3 and i-tRNA are crucial for coupling the movements of NTD and CTD of IF3 during the initiation pathway and in imparting growth fitness to E. coli.

Initiation factor 3 bound to the 30S ribosomal subunit in an initial step of translation.

Bacterial ribosomes require three initiation factors IF1, IF2, and IF3 during the initial steps of translation. These IFs ensure correct base pairing of the initiator tRNA anticodon with the start codon in the mRNA located at the P-site of the 30S ribosomal subunit. IF3 is one of the first IFs to bind to the 30S and plays a crucial role in the selection of the correct start codon and codon: anticodon base pairing. IF3 also prevents the premature association of the 50S subunit of ribosomes and aids in ribosome recycling. IF3 is reported to change binding sites and conformation to ensure translation initiation fidelity. A recent study suggested an initial binding of IF3 CTD away from the P-site and that IF1 and IF2 promote the movement of CTD to the P-site and concomitant movement of NTD. Hence, to visualize the position of IF3 in the absence of any other IFs, we determined cryo-EM structure of the 30S-IF3 complex. The map shows that IF3 is present in an extended conformation with CTD present at the P-site and NTD near the platform even in the absence of IF1 and IF2. Hence, IF3 CTD binds at the P-site and moves away during the accommodation of the initiator tRNA at the P-site in the later steps of translation initiation. Overall, we report the structure of 30S-IF3 which demystifies the starting binding site and conformation of IF3 on the 30S ribosomal subunit.

Hepatitis C prevalence and elimination planning in Pakistan, a bottom-up approach accounting for provincial variation.

In Pakistan, substantial changes to hepatitis C virus (HCV) programming and treatment have occurred since the 2008 nationwide serosurvey estimated a 4.8% anti-HCV prevalence. In the absence of an updated national study, this analysis uses provincial data to estimate a national prevalence and the interventions needed to achieve elimination. Using a Delphi process, epidemiologic HCV data for the four provinces of Pakistan (accounting for 97% of the population) were reviewed with 21 subject-matter experts in Pakistan. Province-level estimates were inputted into a mathematical model to estimate the national HCV disease burden in the absence of intervention (Base), and if the World Health Organization (WHO) elimination targets are achieved by 2030 (80% reduction in new infections, 90% diagnosis coverage, 80% treatment coverage, and 65% reduction in mortality: WHO Elimination). An estimated 9,746,000 (7,573,000-10,006,000) Pakistanis were living with viraemic HCV as of January 1, 2021; a viraemic prevalence of 4.3% (3.3-4.4). WHO Elimination would require an annual average of 18.8 million screens, 1.1 million treatments, and 46,700 new infections prevented anually between 2022 and 2030. Elimination would reduce total infections by 7,045,000, save 152,000 lives and prevent 104,000 incident cases of hepatocellular carcinoma from 2015 to 2030. Blood surveys, programmatic data, and expert panel input uncovered more HCV infections and lower treatment numbers in the provinces than estimated using national extrapolations, demonstrating the benefits of a bottom-up approach. Screening and treatment must increase 20 times and 5 times, respectively, to curb the HCV epidemic in Pakistan and achieve elimination by 2030.

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex.

Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2ß as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

Large-scale movement of eIF3 domains during translation initiation modulate start codon selection.

The eukaryotic initiation factor 3 (eIF3) complex is involved in every step of translation initiation, but there is limited understanding of its molecular functions. Here, we present a single particle electron cryomicroscopy (cryo-EM) reconstruction of yeast 48S ribosomal preinitiation complex (PIC) in an open conformation conducive to scanning, with core subunit eIF3b bound on the 40S interface near the decoding center in contact with the ternary complex eIF2·GTP·initiator tRNA. eIF3b is relocated together with eIF3i from their solvent interface locations observed in other PIC structures, with eIF3i lacking 40S contacts. Re-processing of micrographs of our previous 48S PIC in a closed state also suggests relocation of the entire eIF3b-3i-3g-3a-Cter module during the course of initiation. Genetic analysis indicates that high fidelity initiation depends on eIF3b interactions at the 40S subunit interface that promote the closed PIC conformation, or facilitate the relocation of eIF3b/eIF3i to the solvent interface, on start codon selection.

A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop.

The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

mRNA Recruiting eIF4 Factors Involved in Protein Synthesis and Its Regulation.

The recruitment of mRNA onto the ribosome affects not only global translation but also the spatial and temporal fine-tuning of eukaryotic translation initiation. The eukaryotic initiation factor 4 (eIF4) factors, namely, eIF4G, eIF4E, eIF4A, and eIF4B, facilitate the recruitment of mRNA onto the preassembled 43S pre-initiation complex (PIC), thus leading to the formation of the 48S PIC. Several biochemical and genetic studies have established the roles of eIF4 factors; however, the available structural information is limited. While structures of some of the individual components and subcomplexes are available, the structural details of activated mRNA bound to eIF4 factors (eIF4-mRNA) are missing. Structural characterization of the eIF4-mRNA in association with the 43S PIC in different organisms is crucial for a detailed understanding of mRNA recruitment and for exploiting the structural differences for possible drug design.

Avoidability of drug-induced liver injury (DILI) in an elderly hospital cohort with cases assessed for causality by the updated RUCAM score.

BACKGROUND: Drug-induced liver injury (DILI) represents an increasing morbidity in the general population, but more so in the elderly cohort of patients. Despite this, the concept of its prevention through prospective analysis has largely remained unexamined. We evaluated the utility of recently validated adverse drug reactions (ADR) avoidability tool in a cohort of elderly patients with DILI. METHODS: We examined 38 DILI-drug pairs from n=38 patients in a prospective cohort of patients presenting with adverse drug reactions to a Weill Cornell-affiliated tertiary hospital between February 2019 and January 2020. DILI outcomes were adjudicated by the updated Roussel Uclaf Causality Assessment Method (RUCAM). Two clinical pharmacologists and two general physicians utilized the Liverpool adverse drug reactions avoidability tool (LAAT) and the modified Hallas tools to rate the preventability of DILI-drug pairs. Inter-rater, exact agreement proportions, as well as intraclass correlation coefficients were generated and expressed as ordinal outcomes. RESULTS: The cases examined for the determination of DILI avoidability had probability likelihood of "probable" or "highly probable" by the updated RUCAM scale. Examination of the 38 DILI-drug pairs (n= 38 patients) resulted in a total of 152 ordinal outcome decisions. We found about 32.3% (50/152) and 34.2% (52/152) of DILI-drug pairs were rated as "avoidable" ("probable" or "definite") by the LAAT and the modified Hallas tools respectively. The overall median Krippendorf's kappa with the LAAT was 0.61 (SE 0.12, CI 0.36, 0.85) and for modified Hallas tool was 0.53 (SE 0.18; CI 0.16, 0.89). The inter-rater correlation coefficient (ICC) for the LAAT and modified Hallas were 0.50 [0.32, 0.65] and 0.63 [0.48, 0.76] respectively. Exact pairwise agreement was present in 30/38 (IQR 29.5, 34.5), and 28/38 (IQR 27.5-35.5) of DILI-ADR pairs using the LAAT and modified Hallas tools respectively. CONCLUSION: We found a significant proportion of drug-induced liver injury adjudicated by the updated RUCAM scale in elderly hospitalized cohort of patients were avoidable with significant implication for therapeutic commissioning as well as cost effectiveness interventions in this cohort of patients.

RGG-motif self-association regulates eIF4G-binding translation repressor protein Scd6.

Regulation of mRNA translation plays a key role in the control of gene expression. Scd6, a conserved RGG-motif containing protein represses translation by binding to translation initiation factor eIF4G1. Here we report that Scd6 binds itself in RGG-motif dependent manner and self-association regulates its repression activity. Scd6 self-interaction competes with eIF4G1 binding and methylation of Scd6 RGG-motif by Hmt1 negatively affects self-association. Results pertaining to Sbp1 indicate that self-association could be a general feature of RGG-motif containing translation repressor proteins. Taken together, our study reveals a mechanism of regulation of eIF4G-binding RGG-motif translation repressors.

Turbulence model study for aerodynamic analysis of the leading edge tubercle wing for low Reynolds number flows.

A turbulence model study was performed to analyze the flow around the Tubercle Leading Edge (TLE) wing. Five turbulence models were selected to evaluate aerodynamic force coefficients and flow mechanism by comparing with existing literature results. The selected models are realizable k-ε, k-ω Shear Stress Transport (SST), ( γ - R e θ ) SST model, Transition k-k l -ω model and Stress- ω Reynolds Stress Model (RSM). For that purpose, the TLE wing model was developed by using the NACA0021 airfoil profile. The wing model is designed with tubercle wavelength of 0.11c and amplitude of 0.03c. Numerical simulation was performed at chord-based Reynolds number of Rec = 120,000. The Computational Fluid Dynamic (CFD) simulation reveals that among the selected turbulence models, Stress- ω RSM estimated aerodynamic forces (i.e. lift and drag) coefficients closest to that of the experimental values followed by realizable k-ε, ( γ - R e θ ) SST model, k-ω SST model and k-k l -ω model. However, at a higher angle of attacks i.e. at 16° & 20° k-ω SST model predicted closest drag and lift coefficient to that of the experimental values. Additionally, the critical observation of pressure contour confirmed that at the lower angle of attack Stress- ω RSM predicted strong Leading Edge (LE) suction followed by realizable k-ε, ( γ - R e θ )SST model, k-ω SST model and k-k l -ω model. Thus, the superiority of Stress- ω RSM in predicting the aerodynamic force coefficients is shown by the flow behavior. In addition to this pressure contours also confirmed that k-k l -ω model failed to predict tubercled wing aerodynamic performance. At higher angles of attacks k-ω SST model estimated aerodynamic force coefficients closest to that of the experimental values, thus k-ω SST model is used at 16° & 20° AoAs. The observed streamline behavior for different turbulence models showed that the Stress- ω RSM model and k-k l -ω model failed to model flow behavior at higher AoAs, whereas k-ω SST model is a better approach to model separated flows that experience strong flow recirculation zone.

Cryo-EM structure of wheat ribosome reveals unique features of the plant ribosomes.

Plants being sessile organisms exhibit unique features in ribosomes, which might aid in rapid gene expression and regulation in response to varying environmental conditions. Here, we present high-resolution structures of the 60S and 80S ribosomes from wheat, a monocot staple crop plant (Triticum aestivum). While plant ribosomes have unique plant-specific rRNA modification (Cm1847) in the peptide exit tunnel (PET), the zinc-finger motif in eL34 is absent, and uL4 is extended, making an exclusive interaction network. We note differences in the eL15-helix 11 (25S) interaction, eL6-ES7 assembly, and certain rRNA chemical modifications between monocot and dicot ribosomes. In eukaryotes, we observe highly conserved rRNA modification (Gm75) in 5.8S rRNA and a flipped base (G1506) in PET. These features are likely involved in sensing or stabilizing nascent chain. Finally, we discuss the importance of the universal conservation of three consecutive rRNA modifications in all ribosomes for their interaction with A-site aminoacyl-tRNA.

Railway track surface faults dataset.

Railway infrastructure maintenance is critical for ensuring safe and efficient transportation networks. Railway track surface defects such as cracks, flakings, joints, spallings, shellings, squats, grooves pose substantial challenges to the integrity and longevity of the tracks. To address these challenges and facilitate further research, a novel dataset of railway track surface faults has been presented in this paper. It is collected using the EKENH9R cameras mounted on a railway inspection vehicle. This dataset represents a valuable resource for the railway maintenance and computer vision related scientific communities. This dataset includes a diverse range of real-world track surface faults under various environmental conditions and lighting scenarios. This makes it an important asset for the development and evaluation of Machine Learning (ML), Deep Learning (DL), and image processing algorithms. This paper also provides detailed annotations and metadata for each image class, enabling precise fault classification and severity assessment of the defects. Furthermore, this paper discusses the data collection process, highlights the significance of railway track maintenance, emphasizes the potential applications of this dataset in fault identification and predictive maintenance, and development of automated inspection systems. We encourage the research community to utilize this dataset for advancing the state-of-the-art research related to railway track surface condition monitoring.

Mechanistic insights into cognate substrate discrimination during proofreading in translation.

Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "Double-Sieve Model." Strikingly using solution based direct binding studies, NMR-heteronuclear single quantum coherence (HSQC) and isothermal titration calorimetry experiments, with an editing domain of threonyl-tRNA synthetase, we show that the cognate substrate can gain access and bind to the editing pocket. High-resolution crystal structural analyses reveal that functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A strategically positioned "catalytic water" molecule is excluded to avoid hydrolysis of the cognate substrate using a "RNA mediated substrate-assisted catalysis mechanism" at the editing site. The mechanistic proof of the critical role of RNA in proofreading activity is a completely unique solution to the problem of cognate-noncognate selection mechanism.

Selection of start codon during mRNA scanning in eukaryotic translation initiation.

Accurate and high-speed scanning and subsequent selection of the correct start codon are important events in protein synthesis. Eukaryotic mRNAs have long 5' UTRs that are inspected for the presence of a start codon by the ribosomal 48S pre-initiation complex (PIC). However, the conformational state of the 48S PIC required for inspecting every codon is not clearly understood. Here, atomistic molecular dynamics (MD) simulations and energy calculations suggest that the scanning conformation of 48S PIC may reject all but 4 (GUG, CUG, UUG and ACG) of the 63 non-AUG codons, and initiation factor eIF1 is crucial for this discrimination. We provide insights into the possible role of initiation factors eIF1, eIF1A, eIF2α and eIF2ß in scanning. Overall, the study highlights how the scanning conformation of ribosomal 48S PIC acts as a coarse selectivity checkpoint for start codon selection and scans long 5' UTRs in eukaryotic mRNAs with accuracy and high speed.

Triaxial bearing vibration dataset of induction motor under varying load conditions.

Rotating machines as core component of an industry can effectively be monitored through vibration analysis. Considering the importance of vibration in industrial condition monitoring, this article reports and discusses triaxial vibration data for motor bearing faults detection and identification. The data is acquired using a MEMS based triaxial accelerometer and the National Instruments myRIO board. The bearing conditions include healthy bearing, bearings with inner race faults, and bearings with outer race faults. For each faulty bearing condition, the three-phase induction motor is operated under three different load conditions. The dataset can be used to assess performance of newly developed methods for effective bearing fault diagnosis. Mendeley Data. http://dx.doi.org/10.17632/fm6xzxnf36.2.

Evidence for low-level translation in human erythrocytes.

It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labeling, and RiboPuromycylation. [35S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that HBA (α-globin) and HBB (ß-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and ß-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2.

The SARS-CoV-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-CoV-2 bind in the mRNA entry channel of the 40S ribosomal subunit and blocks mRNA entry, thereby shutting down host protein synthesis. Nsp1 suppresses host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter. Among the top hits obtained, montelukast sodium hydrate binds to Nsp1 with a binding affinity (KD) of 10.8 ± 0.2 µM in vitro. It forms a stable complex with Nsp1-C-ter in simulation runs with -95.8 ± 13.3 kJ/mol binding energy. Montelukast sodium hydrate also rescues the inhibitory effect of Nsp1 in host protein synthesis, as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, it shows antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We, therefore, propose montelukast sodium hydrate can be used as a lead molecule to design potent inhibitors to help combat SARS-CoV-2 infection.