Biology subjects

Sharma, H.

Publications and source records attributed to Sharma, H..

36 records · Page 2Linked to original sources

bioRxiv2024

Genome-Wide Identification of G-Quadruplex forming regions in Arabidopsis: Unraveling the Role of Pif like Helicase (AtPLH1) in Gene Regulation and Stress Response

G-quadruplexes (GQSes) are highly stable DNA secondary structures, which exist as knots in the genome during different cellular processes like replication, transcription and translation. Although several studies have shown the role of GQS-helicases regulating several cellular processes in yeast and human, their detailed characterization in plants is still lacking. In this study, we identified GQS-enriched regions by DNA affinity purification followed by sequencing from Arabidopsis Pif-like helicase 1 (Atplh1) mutant. Differentially enriched peaks (DEPs) in the mutant showed preferential distribution in the exonic and promoter regions. The genes involved in various processes like transcriptional regulation, UDP- glucosylation, response to abiotic stress, ethylene biosynthesis and response to carbohydrate stimulus, were found to be differentially regulated between control and mutant plants. The differentially expressed genes (DEGs) showed enrichment of binding sites of ERF, WRKY, BBM and BIM transcription factors. Further, the DEGs harboring DEPs were found associated with response to wounding and salt stress, response to unfolded protein, heat stress response and UDP-glycosyl transferase activity. In addition, the mutants exhibited lesser growth inhibition under cold stress. Overall, our study identified genome-wide GQSes in Arabidopsis and altered gene expression regulated by AtPLH1.

plant biology↗

bioRxiv2023

In vitro reconstitution reveals membrane clustering and double-stranded RNA recruitment by the enteroviral AAA+ ATPase 2C

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2Cs association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divides the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

microbiology↗

bioRxiv2023

Kinase inhibitor-induced cell-type specific vacuole formation in the absence of canonical ATG5-dependent autophagy

Pyridinyl imidazole class p38 MAPK/{beta} (MAPK14/MAPK11) inhibitors including SB202190 have been shown to induce a cell-type specific defective autophagy response resulting in micron-scale vacuole formation, autophagy-dependent death, and tumor growth suppression in vivo. We had earlier shown that this is an off-target effect of SB202190. Here we provide evidence that the cell-type specific vacuole formation is independent of canonical autophagy pathway. While SB202190 seems to interfere with autophagic flux in many cell lines in parallel to vacuolation, autophagy-deficient DU-145 cells and CRISPR/Cas9 gene-edited ATG5 knockout A549 cells also undergo vacuolation upon SB202190 treatment. Late-endosomal GTPase RAB7 colocalizes with these compartments and RAB7 GTP-binding seems to be essential for SB202190-induced vacuolation. RAB7 is a driver of tumor progression and interfering with RAB7-positive endo/lysosomal compartments may enhance cytotoxicity. A screen for modulators of SB202190-induced vacuolation revealed molecules including multi-kinase inhibitor Sorafenib as inhibitor of vacuolation and sorafenib co-treatment enhanced the cytotoxicity of SB202190. Moreover VE-821, an ATR kinase inhibitor was found to phenocopy the cell-type specific vacuolation response of SB202190. To identify the factors determining the cell-type specificity of the vacuolation response induced by SB-compounds and VE-821, we compared the transcriptomics data from vacuole forming and non-vacuole forming cancer cell lines and identified a gene expression signature which may define sensitivity of cancer cells to these small-molecule kinase inhibitors. Further analyses using the small molecule tools and the gene signature discovered here, could reveal novel mechanisms regulating this interesting phenotype relevant to anti-cancer therapy.

cell biology↗

bioRxiv2023

Functional annotation of a divergent genome using sequence and structure-based homology.

BackgroundMicrosporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence homology. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. ResultsIn this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin, allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted open reading frames, of which 89 % were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. ConclusionWe provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

genomics↗

bioRxiv2023

Ribosome clustering and surface layer reorganization in the microsporidian host-invasion apparatus.

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and demonstrates that ribosomes are efficiently transported through polar tubes in a spiral-like parallel arrangement.

biophysics↗

bioRxiv2023

Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA-RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

molecular biology↗

bioRxiv2023

A Robust Deep Learning Approach for Joint Nuclei Detection and Cell Classification in Pan-Cancer Histology Images

Advanced image processing methods have shown promise in computational pathology, including the extraction of crucial microscopic features from histology images. Accurate detection and classification of cell nuclei from whole-slide images (WSI) play a crucial role in capturing the molecular and morphological landscape of the tissue sample. They enable widespread downstream applications, including cancer diagnosis, prognosis, and discovery of novel markers. Robust nuclei detection and classification are challenging due to the high intra-class variability and inter-class similarity of the microscopic morphological features. This is further compounded by the domain shift arising due to the variability in tissue types, staining protocols, and image acquisition. Motivated by the ability of the recent deep learning techniques to learn complex patterns in a biasfree manner, we develop a novel and robust deep learning model TransNuc, based on vision transformers, for simultaneous detection and classification of cell nuclei from H&E stained WSI. We benchmarked TransNuc on the comprehensive Open Pan-cancer Histology Dataset (PanNuke), sampled from over 20,000 WSI, comprising 19 different tissue types and five clinically important cell classes, namely, Neoplastic, Epithelial, Inflammatory, Connective, and Dead cells. TransNuc exhibited superior performance compared to the state-of-theart, including Hover-Net and Micro-Net. TransNuc was able to learn robust feature representations and thereby perform consistently better for the abundant classes such as neoplastic, and the under-represented classes such as dead cells. Similar performance gains were also obtained for epithelial and connective classes that have a significant inter-class morphological similarity.

pathology↗

bioRxiv2023

Development of Drosophila as a metazoan model to study inorganic polyphosphate biology

Polyphosphate (polyP) exists in all life forms; however, its biological functions in metazoans are understudied. Here, we explored Drosophila, to our knowledge, as the first genetic model to explore polyP biology in metazoans. We established biochemical and in situ methods to detect, quantify, and visualise polyP in Drosophila. We then engineered a FLYX system to deplete polyP in subcellular compartments in a tissue-specific manner. Using these tools, we demonstrated a spatiotemporal and subcellular compartment-specific regulation of polyP levels in various developmental stages and tissue types. We discovered that polyP is crucial for Drosophila hemolymph clotting and proper developmental timing, consistent with an evolutionarily conserved role as exogenous polyP also accelerates mammalian blood clotting. Further, the transcriptomics analysis of polyP-depleted larvae demonstrates the impact of polyP on several cellular processes, including translation. These observations underscore the utility of the toolkit we developed to discover previously unknown polyP functions in metazoans.

cell biology↗

bioRxiv2022

Efficient recall of Omicron-reactive B cell memory after a third dose of SARS-CoV-2 mRNA vaccine

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for [~]9-10 months after the primary 2-dose mRNA vaccine series, as well as for [~]3 months after a 3rd mRNA vaccine dose. Notably, antibody decay slowed significantly between 6- and 9-months post-primary vaccination, essentially stabilizing at the time of the 3rd dose. Antibody quality also continued to improve for at least 9 months after primary 2-dose vaccination. Spike- and RBD-specific memory B cells were stable through 9 months post-vaccination with no evidence of decline over time, and [~]40-50% of RBD-specific memory B cells were capable of simultaneously recognizing the Alpha, Beta, Delta, and Omicron variants. Omicron-binding memory B cells induced by the first 2 doses of mRNA vaccine were boosted significantly by a 3rd dose and the magnitude of this boosting was similar to memory B cells specific for other variants. Pre-3rd dose memory B cell frequencies correlated with the increase in neutralizing antibody titers after the 3rd dose. In contrast, pre-3rd dose antibody titers inversely correlated with the fold-change of antibody boosting, suggesting that high levels of circulating antibodies may limit reactivation of immunological memory and constrain further antibody boosting by mRNA vaccines. These data provide a deeper understanding of how the quantity and quality of antibody and memory B cell responses change over time and number of antigen exposures. These data also provide insight into potential immune dynamics following recall responses to additional vaccine doses or post-vaccination infections. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/481163v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@123d2d9org.highwire.dtl.DTLVardef@e7db82org.highwire.dtl.DTLVardef@1fc73deorg.highwire.dtl.DTLVardef@11b21f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Insights into the Human Gut Virome by Sampling a Population from the Indian Subcontinent

Gut virome plays an important role in human physiology but remains poorly understood. This study reports an investigation of the human gut DNA-virome of a previously unexplored ethnic population through metagenomics of faecal samples collected from individuals residing in Northern India. Analysis shows that, similar to the populations investigated earlier, majority of the identified virome belongs to bacteriophages and a smaller fraction (< 20%) consists of viruses that infect animals, archaea, protists, multiple domains or plants. However, crAss-like phages, in this population, are dominated by the genera VII, VIII and VI. Interestingly, it also reveals the presence of a virus family, Sphaerolipoviridae, which has not been detected in the human gut earlier. Viral families, Siphoviridae, Myoviridae, Podoviridae, Microviridae, Herelleviridae and Phycodnaviridae are detected in all of the analyzed individuals, which supports the existence of a core virome. Lysogeny-associated genes were found in less than 10% of the assembled genomes and a negative correlation was observed in the richness of bacterial and free-viral species, suggesting that the dominant lifestyle of gut phage is not lysogenic. This is in contrast to some of the earlier studies. Further, several hundred high-quality viral genomes were recovered. Detailed characterization of these genomes would be useful for understanding the biology of these viruses and their significance in human physiology. ImportanceViruses are important constituents of the human gut microbiome but it remains poorly understood. The Indian subcontinent is a unique biogeographic region and the Indian population is known to harbour a distinct bacterial microbiome. However, the gut virome in this population has not been investigated earlier. Therefore, in this study, we investigated fecal samples of 12 healthy individuals to analyze their gut virome, through metagenomics.

microbiology↗

bioRxiv2021

mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naive and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=146 HEIGHT=200 SRC="FIGDIR/small/457229v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@16c64b1org.highwire.dtl.DTLVardef@146ca3aorg.highwire.dtl.DTLVardef@86b7edorg.highwire.dtl.DTLVardef@956879_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Perturbation of ribosomal subunits dynamics by inhibitors of tRNA translocation

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA translocation), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, EF-G-induced transition to the rotated/swiveled state of the SSU is hardly affected. The major effect of the antibiotics is observed at the stage when the SSU body and the head domain move backward. Vio, Spc and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

molecular biology↗

bioRxiv2020

Hemoglobin drives inflammation and initiates antigen spread and nephritis in lupus

Hemoglobin (Hb) has well-documented inflammatory effects and is normally efficiently scavenged; clearance mechanisms can be overwhelmed during conditions of erythrocyte lysis, a condition that may occur in systemic lupus erythematosus. Whether Hb is preferentially inflammatory in lupus and additionally induces autoreactivity against prominent autoantigens was assessed. Peripheral blood mononuclear cells derived from SLE patients secreted higher levels of lupus-associated inflammatory cytokines when incubated with Hb, effects negated by haptoglobin. Hb (more particularly, ferric Hb) triggered the preferential release of lupus-associated cytokines from splenocytes, B cells, CD4 T cells, CD8 T cells and plasmacytoid dendritic cells isolated from aging NZM2410 mice, and also had mitogenic effects on B cells. Ferric Hb activated multiple signaling pathways which were differentially responsible for the generation of specific cytokines; inflammatory signaling also appeared to be cell-context dependent. Pull-downs, followed by mass spectrometry, revealed interactions of Hb with several lupus-associated autoantigens; co-incubation of ferric Hb with apoptotic blebs (structures which contain packaged autoantigens, believed to trigger lupus autoreactivity) revealed synergies (in terms of cytokine release and autoantibody production in vitro) that were also restricted to the lupus genotype. Infusion of ferric Hb into NZM2410 mice led to enhanced release of lupus-associated cytokines, the generation of a spectrum of autoantibodies, and enhanced-onset glomerulosclerosis. Given that the biased recognition of ferric Hb in a lupus milieu, in concert with lupus-associated autoantigens, elicits the generation of inflammatory cytokines from multiple immune cell types and stimulates the generation of potentially pathogenic autoantibodies, neutralization of Hb could have beneficial effects.

immunology↗

bioRxiv2020

Thaumatin-like proteins: Molecular characterization, evolutionary analysis and expression profiling in major cereal crops

Thaumatin-like proteins (TLPs) are related to the pathogenesis-related-5 (PR-5) family and involved in stress response. Herein, a total of 93 TLP genes were identified in the genome of Triticum aestivum. Further, we identified 26, 27, 39 and 37 TLP genes in the Brachypodium distachyon, Oryza sativa, Sorghum bicolor and Zea mays genomes for comparative characterization, respectively. They could be grouped into small and long TLPs with conserved thaumatin signature motif. Tightly clustered genes exhibited conserved gene and protein structure. The physicochemical analyses suggested significant differences between small and long TLPs. Evolutionary analyses suggested the role of duplication events and purifying selection in the expansion of the TLP gene family. Expression analyses revealed the possible roles of TLPs in plant development and abiotic and fungal stress response. Recombinant expression of TaTLP2-B in Saccharomyces cerevisiae provided significant tolerance against cold, heat, drought and salt stresses. The results depicted the importance of TLPs in cereal crops that would be highly useful in future crop improvement programs.

genomics↗

bioRxiv2020

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-EM structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are universally highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which acts as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

microbiology↗

bioRxiv2020

Gradient of Tactile Properties in the Rat Whisker Pad

The array of vibrissae on a rats face is the first stage in a high-resolution tactile sensing system. Progressing from rostral to caudal in any vibrissae row results in an increase in whisker length and thickness. This in turn may provide a systematic map of separate tactile channels governed by the mechanical properties of the whiskers. To examine whether this map is expressed in a location-dependent transformation of tactile signals into whisker vibrations and neuronal responses, we monitored whiskers movements across various surfaces and edges. We found a robust rostral-caudal gradient of tactile information transmission in which rostral shorter vibrissae displayed a higher sensitivity and a bigger differences in response to the different textures, whereas longer caudal vibrissae were less sensitive to the different textures. Nonetheless, we found that texture identity is not represented spatially across the whisker pad. Based on the responses of first-order sensory neurons, we found that neurons innervating rostral whiskers were better suited for textures discrimination, whereas neurons innervating caudal whiskers were more suited for edge detection. To examine the functional role of this organization, we monitored the whisking activity of awake rats foraging for food. We found a caudal-rostral gradient of whisking angle movements in which the longer caudal vibrissae spanned a larger space making them more suitable for object localization, whereas the rostral shorter vibrissae hardly moved. These results suggest that the whisker array in rodents forms a sensory structure in which different facets of tactile information are transmitted through location-dependent gradient of vibrissae on the rats face.

neuroscience↗

bioRxiv2018

Assembly Defects Abrogate Proofreading by Initiation Factors and License the Entry of Premature Ribosomes into the Translation Cycle

Numerous quality control steps are deployed by the translational machinery to ensure faithful decoding of genetic message to synthesize proteins. However, what transpires to quality control mechanism during protein synthesis when the ribosomes are produced with assembly defects remains enigmatic. In E. coli, we show that ribosomes with assembly defects evade the proofreading steps during translation initiation and participate in the translation cycle. Such ribosomes show severely compromised decoding capabilities that give rise to errors in initiation and elongation. Tracing the genesis, we discovered that the assembly defects compromise the binding of initiation factors, thus licensing the rapid transitioning of 30S (pre) initiation complex to 70S initiation complex by tempering the proofreading mechanism. Overall, our work highlights that a mass balance deficit between premature ribosomes and initiation factors steers the entry of premature ribosomes into the translation cycle.

biochemistry↗

bioRxiv2018

Dynamics of ribosomes and release factors during translation termination in E. coli

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET together with ensemble kinetics, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, while each factor alone has its distinct signature on the ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. GTP cleavage helps RF3 release from ribosomes stalled in the rotated state in the absence of RF1. Our data suggest how the stochastic assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

biochemistry↗