Ribosome Recycling Factor Research Articles

Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome

BackgroundDeoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing.ResultsThe co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins.ConclusionThe results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

CLUH maintains functional mitochondria and translation in motoneuronal axons and prevents peripheral neuropathy.

Transporting and translating mRNAs in axons is crucial for neuronal viability. Local synthesis of nuclear-encoded mitochondrial proteins protects long-lived axonal mitochondria from damage; however, the regulatory factors involved are largely unknown. We show that CLUH, which binds mRNAs encoding mitochondrial proteins, prevents peripheral neuropathy and motor deficits in the mouse. CLUH is enriched in the growth cone of developing spinal motoneurons and is required for their growth. The lack of CLUH affects the abundance of target mRNAs and the corresponding mitochondrial proteins more prominently in axons, leading to ATP deficits in the growth cone. CLUH interacts with ribosomal subunits, translation initiation, and ribosome recycling components and preserves axonal translation. Overexpression of the ribosome recycling factor ABCE1 rescues the mRNA and translation defects, as well as the growth cone size, in CLUH-deficient motoneurons. Thus, we demonstrate a role for CLUH in mitochondrial quality control and translational regulation in axons, which is essential for their development and long-term integrity and function.

Genetic analysis of translation initiation in bacteria: An initiator tRNA-centric view.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination, and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N10 -formyl-tetrahydrofolate (N10 -fTHF), respectively. Both methionine and N10 -fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. The acceptor stem region is important in formylation of the amino acid attached to i-tRNA and in its initial binding to the P-site. And, the 3GC pairs are crucial in transiting the i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor, and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

Identification of virulence-associated factors in Vibrio parahaemolyticus with special reference to moonlighting protein: a secretomics study.

Vibrio parahaemolyticus causes seafood-borne gastroenteritis infection in human which can even lead to death. The pathogenic strain of V. parahaemolyticus secretes different types of virulence factors thatare directly injected into the host cell by a different type of secretion system which helps bacteria to establish its own ecological niche within the organism. Therefore, the aim of this study was to isolate the extracellular secreted proteins from the trh positive strain of V. parahaemolyticus and identify them using two-dimensional gel electrophoresis and MALDI-TOFMS/MS. Seventeen different cellular proteins viz, Carbamoyl-phosphate synthase, 5-methyltetrahydropteroyltriglutamate, tRNA-dihydrouridine synthase, Glycerol-3-phosphate dehydrogenase, Orotidine 5'-phosphate decarboxylase, Molybdenum import ATP-binding protein, DnaJ, DNA polymerase IV, Ribosomal RNA small subunit methyltransferase G, ATP synthase subunit delta and gamma, Ribosome-recycling factor, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, tRNA pseudouridine synthase B, Ditrans, polycis-undecaprenyl-diphosphate synthase, Oxygen-dependent coproporphyrinogen-III oxidase, and Peptide deformylase 2 were identified whichare mainly involved in different metabolic and biosynthetic pathways. Furthermore, the molecular function of the identified proteins were associated with catalytic activity, ligase activity, transporter, metal binding, and ATP synthase when they are intercellular. However, to understand the importance of these secreted proteins in the infection and survival of bacteria inside the host cell, pathogen-host protein-protein interactions (PPIs) were carried out which identified the association of eight secreted proteins with 41 human proteins involved in different cellular pathways, including ubiquitination degradation, adhesion, inflammation, immunity, and programmed cell death. The present study provides unreported strategies on host-cell environment's survival and adaptation mechanisms for the successful establishment of infections and intracellular propagation.

Role of ribosome recycling factor in natural termination and translational coupling as a ribosome releasing factor.

The role of ribosome recycling factor (RRF) of E. coli was studied in vivo and in vitro. We used the translational coupling without the Shine-Dalgarno sequence of downstream ORF (d-ORF) as a model system of the RRF action in natural termination of protein synthesis. For the in vivo studies we used the translational coupling by the adjacent coat and lysis genes of RNA phage GA sharing the termination and initiation (UAAUG) and temperature sensitive RRF. The d-ORF translation was measured by the expression of the reporter lacZ gene connected to the 5'-terminal part of the lysis gene. The results showed that more ribosomes which finished upstream ORF (u-ORF) reading were used for downstream reading when RRF was inactivated. The in vitro translational coupling studies with 027mRNA having the junction sequence UAAUG with wild-type RRF were carried out with measuring amino acids incorporation. The results showed that ribosomes released by RRF read downstream from AUG of UAAUG. In the absence of RRF, ribosomes read downstream in frame with UAA. These in vivo and in vitro studies indicate that RRF releases ribosomes from mRNA at the termination codon of u-ORF. Furthermore, the non-dissociable ribosomes read downstream from AUG of UAAUG with RRF in vitro. This suggests that complete ribosomal splitting is not required for ribosome release by RRF in translational coupling. The data are consistent with the interpretation that RRF functions mostly as a ribosome releasing factor rather than ribosome splitting factor. Additionally, the in vivo studies showed that short (less than 5 codons) u-ORF inhibited d-ORF reading by ribosomes finishing u-ORF reading, suggesting that the termination process in short ORF is not similar to that in normal ORF. This means that all the preexisting studies on RRF with short mRNA may not represent what goes on in natural termination step.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Comparing proteome changes involved in biofilm formation by Streptococcus mutans after exposure to sucrose and starch.

Streptococcus mutans is a main organism of tooth infections including tooth decay and periodontitis. The aim of this study was to assess the influence of sucrose and starch on biofilm formation and proteome profile of S. mutans ATCC 35668 strain. The biofilm formation was assessed by microtiter plating method. Changes in bacterial proteins after exposure to sucrose and starch carbohydrates were analyzed using matrix-assisted laser desorption/ionization mass spectrometry. The biofilm formation of S. mutans was increased to 391.76% in 1% sucrose concentration, 165.76% in 1% starch, and 264.27% in the 0.5% sucrose plus 0.5% starch in comparison to biofilm formation in the media without sugars. The abundance of glutamines, adenylate kinase, and 50S ribosomal protein L29 was increased under exposure to sucrose. Upregulation of lactate utilization protein C, 5-hydroxybenzimidazole synthase BzaA, and 50S ribosomal protein L16 was formed under starch exposure. Ribosome-recycling factor, peptide chain release factor 1, and peptide methionine sulfoxide reductase MsrB were upregulated under exposure to sucrose in combination with starch. The results demonstrated that the carbohydrates increase microbial pathogenicity. In addition, sucrose and starch carbohydrates can induce biofilm formation of S. mutans via various mechanisms such as changes in the expression of special proteins.

Effects of Sub-Minimum Inhibitory Concentrations of Imipenem and Colistin on Expression of Biofilm-Specific Antibiotic Resistance and Virulence Genes in Acinetobacter baumannii Sequence Type 1894.

Antibiotics at suboptimal doses promote biofilm formation and the development of antibiotic resistance. The underlying molecular mechanisms, however, were not investigated. Here, we report the effects of sub-minimum inhibitory concentrations (sub-MICs) of imipenem and colistin on genes associated with biofilm formation and biofilm-specific antibiotic resistance in a multidrug-tolerant clinical strain of Acinetobacter baumannii Sequence Type (ST) 1894. Comparative transcriptome analysis was performed in untreated biofilm and biofilm treated with sub-MIC doses of imipenem and colistin. RNA sequencing data showed that 78 and 285 genes were differentially expressed in imipenem and colistin-treated biofilm cells, respectively. Among the differentially expressed genes (DEGs), 48 and 197 genes were upregulated exclusively in imipenem and colistin-treated biofilm cells, respectively. The upregulated genes included those encoding matrix synthesis (pgaB), multidrug efflux pump (novel00738), fimbrial proteins, and homoserine lactone synthase (AbaI). Upregulation of biofilm-associated genes might enhance biofilm formation when treated with sub-MICs of antibiotics. The downregulated genes include those encoding DNA gyrase (novel00171), 30S ribosomal protein S20 (novel00584), and ribosome releasing factor (RRF) were downregulated when the biofilm cells were treated with imipenem and colistin. Downregulation of these genes affects protein synthesis, which in turn slows down cell metabolism and makes biofilm cells more tolerant to antibiotics. In this investigation, we also found that 5 of 138 small RNAs (sRNAs) were differentially expressed in biofilm regardless of antibiotic treatment or not. Of these, sRNA00203 showed the highest expression levels in biofilm. sRNAs regulate gene expression and are associated with biofilm formation, which may in turn affect the expression of biofilm-specific antibiotic resistance. In summary, when biofilm cells were exposed to sub-MIC doses of colistin and imipenem, coordinated gene responses result in increased biofilm production, multidrug efflux pump expression, and the slowdown of metabolism, which leads to drug tolerance in biofilm. Targeting antibiotic-induced or repressed biofilm-specific genes represents a new strategy for the development of innovative and effective treatments for biofilm-associated infections caused by A. baumannii.

Prevention of ribosome collision-induced neuromuscular degeneration by SARS CoV-2–encoded Nsp1

An overarching goal of aging and age-related neurodegenerative disease research is to discover effective therapeutic strategies applicable to a broad spectrum of neurodegenerative diseases. Little is known about the extent to which targetable pathogenic mechanisms are shared among these seemingly diverse diseases. Translational control is critical for maintaining proteostasis during aging. Gaining control of the translation machinery is also crucial in the battle between viruses and their hosts. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing COVID-19 pandemic. Here, we show that overexpression of SARS-CoV-2-encoded nonstructural protein 1 (Nsp1) robustly rescued neuromuscular degeneration and behavioral phenotypes in Drosophila models of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. These diseases share a common mechanism: the accumulation of aberrant protein species due to the stalling and collision of translating ribosomes, leading to proteostasis failure. Our genetic and biochemical analyses revealed that Nsp1 acted in a multipronged manner to resolve collided ribosomes, abort stalled translation, and remove faulty translation products causative of disease in these models, at least in part through the ribosome recycling factor ABCE1, ribosome-associated quality-control factors, autophagy, and AKT signaling. Nsp1 exhibited exquisite specificity in its action, as it did not modify other neurodegenerative conditions not known to be associated with ribosome stalling. These findings uncover a previously unrecognized mechanism of Nsp1 in manipulating host translation, which can be leveraged for combating age-related neurodegenerative diseases that are affecting millions of people worldwide and currently without effective treatment.

Human ABCE1 exhibits temperature-dependent heterologous co-functionality in S. cerevisiae.

ABCE1 protein (Rli1 in Saccharomyces cerevisiae) is a unique ribosome recycling factor that is composed of an N‐terminal FeS cluster domain and two ATPase domains. Here, we report that heterologous expression of human ABCE1 in S. cerevisiae is unable to complement conditional knockout of ABCE1 (Rli1), at a typical experimental temperature of 30 °C. However, low but significant growth was observed at high temperature, 37 °C. Considering the close interaction of ABCE1 with translation factors and ribosomal components, the observed temperature‐dependent complementation may be attributed to heterologous co‐functionality of ABCE1 with S. cerevisiae factor(s), and might reflect functional upregulation of human ABCE1 at its functional temperature.

Genomic selection and genetic architecture of agronomic traits during modern rapeseed breeding.

Rapeseed (Brassica napus L.) is an important oil-producing crop for the world. Its adaptation, yield and quality have been considerably improved in recent decades, but the genomic basis underlying successful breeding selection remains unclear. Hence, we conducted a comprehensive genomic assessment of rapeseed in the breeding process based on the whole-genome resequencing of 418 diverse rapeseed accessions. We unraveled the genomic basis for the selection of adaptation and agronomic traits. Genome-wide association studies identified 628 associated loci-related causative candidate genes for 56 agronomically important traits, including plant architecture and yield traits. Furthermore, we uncovered nonsynonymous mutations in plausible candidate genes for agronomic traits with significant differences in allele frequency distributions across the improvement process, including the ribosome recycling factor (BnRRF) gene for seed weight. This study provides insights into the genomic basis for improving rapeseed varieties and a valuable genomic resource for genome-assisted rapeseed breeding.

Systematic evolution of initiation factor 3 and the ribosomal protein uS12 optimizes Escherichia coli growth with an unconventional initiator tRNA

The anticodon stem of initiator tRNA (i-tRNA) possesses the characteristic three consecutive GC base pairs (G29:C41, G30:C40, and G31:C39 abbreviated as GC/GC/GC or 3GC pairs) crucial to commencing translation. To understand the importance of this highly conserved element, we isolated two fast-growing suppressors of Escherichia coli sustained solely on an unconventional i-tRNA (i-tRNAcg/GC/cg ) having cg/GC/cg sequence instead of the conventional GC/GC/GC. Both suppressors have the common mutation of V93A in initiation factor 3 (IF3), and additional mutations of either V32L (Sup-1) or H76L (Sup-2) in small subunit ribosomal protein 12 (uS12). The V93A mutation in IF3 was necessary for relaxed fidelity of i-tRNA selection to sustain on i-tRNAcg/GC/cg though with a retarded growth. Subsequent mutations in uS12 salvaged the retarded growth by enhancing the fidelity of translation. The H76L mutation in uS12 showed better fidelity of i-tRNA selection. However, the V32L mutation compensated for the deficient fidelity of i-tRNA selection by ensuring an efficient fidelity check by ribosome recycling factor (RRF). We reveal unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, elongation factor-G (EF-G), RRF, and Pth (peptidyl-tRNA hydrolase) which, taken together, govern the fidelity of translation in bacteria.

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein.

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

Structural basis of translation termination, rescue, and recycling in mammalian mitochondria

The mitochondrial translation system originates from a bacterial ancestor but has substantially diverged in the course of evolution. Here, we use single-particle cryo-electron microscopy (cryo-EM) as a screening tool to identify mitochondrial translation termination mechanisms and to describe them in molecular detail. We show how mitochondrial release factor 1a releases the nascent chain from the ribosome when it encounters the canonical stop codons UAA and UAG. Furthermore, we define how the peptidyl-tRNA hydrolase ICT1 acts as a rescue factor on mitoribosomes that have stalled on truncated messages to recover them for protein synthesis. Finally, we present structural models detailing the process of mitochondrial ribosome recycling to explain how a dedicated elongation factor, mitochondrial EFG2 (mtEFG2), has specialized for cooperation with the mitochondrial ribosome recycling factor to dissociate the mitoribosomal subunits at the end of the translation process.

Specialized Metabolites from Ribosome Engineered Strains of Streptomyces clavuligerus.

Bacterial specialized metabolites are of immense importance because of their medicinal, industrial, and agricultural applications. Streptomyces clavuligerus is a known producer of such compounds; however, much of its metabolic potential remains unknown, as many associated biosynthetic gene clusters are silent or expressed at low levels. The overexpression of ribosome recycling factor (frr) and ribosome engineering (induced rpsL mutations) in other Streptomyces spp. has been reported to increase the production of known specialized metabolites. Therefore, we used an overexpression strategy in combination with untargeted metabolomics, molecular networking, and in silico analysis to annotate 28 metabolites in the current study, which have not been reported previously in S. clavuligerus. Many of the newly described metabolites are commonly found in plants, further alluding to the ability of S. clavuligerus to produce such compounds under specific conditions. In addition, the manipulation of frr and rpsL led to different metabolite production profiles in most cases. Known and putative gene clusters associated with the production of the observed compounds are also discussed. This work suggests that the combination of traditional strain engineering and recently developed metabolomics technologies together can provide rapid and cost-effective strategies to further speed up the discovery of novel natural products.

In silico analysis of bacterial translation factors reveal distinct translation event specific pI values

BackgroundProtein synthesis is a cellular process that takes place through the successive translation events within the ribosome by the event-specific protein factors, namely, initiation, elongation, release, and recycling factors. In this regard, we asked the question about how similar are those translation factors to each other from a wide variety of bacteria? Hence, we did a thorough in silico study of the translation factors from 495 bacterial sp., and 4262 amino acid sequences by theoretically measuring their pI and MW values that are two determining factors for distinguishing individual proteins in 2D gel electrophoresis in experimental procedures. Then we analyzed the output from various angles.ResultsOur study revealed the fact that it’s not all same, or all random, but there are distinct orders and the pI values of translation factors are translation event specific. We found that the translation initiation factors are mainly basic, whereas, elongation and release factors that interact with the inter-subunit space of the intact 70S ribosome during translation are strictly acidic across bacterial sp. These acidic elongation factors and release factors contain higher frequencies of glutamic acids. However, among all the translation factors, the translation initiation factor 2 (IF2) and ribosome recycling factor (RRF) showed variable pI values that are linked to the order of phylogeny.ConclusionsFrom the results of our study, we conclude that among all the bacterial translation factors, elongation and release factors are more conserved in terms of their pI values in comparison to initiation and recycling factors. Acidic properties of these factors are independent of habitat, nature, and phylogeny of the bacterial species. Furthermore, irrespective of the different shapes, sizes, and functions of the elongation and release factors, possession of the strictly acidic pI values of these translation factors all over the domain Bacteria indicates that the acidic nature of these factors is a necessary criterion, perhaps to interact into the partially enclosed rRNA rich inter-subunit space of the translating 70S ribosome.

Differential expression of proteins in Pseudomonas mendocina SMSKVR-3 under arsenate stress.

This study focuses on analyzing the protein expression pattern of intracellular proteins when Pseudomonas mendocina SMSKVR-3 exposed to 300 mM of arsenate to find out the proteins that are overexpressed or exclusively expressed in response to arsenate. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein expression at different time intervals showed the highest number of protein bands (14) that are overexpressed at 8 h of the time interval. It was also observed that treatment with at least 200 mM of As(V) is required to induce a difference in protein expression. Two-dimensional (2D)-PAGE analysis of 8-h sample exhibited 146 unique spots, 45 underexpressed, and 46 overexpressed spots in arsenate-treated sample. Based on the highest percent volume and fold change, three unique spots and one overexpressed spot were selected and analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry (MS) analysis followed by the MASCOT search. These proteins were identified as ribosome-recycling factor (20.13 kDa), polyphosphate:ADP/GDP phosphotransferase (40.88 kDa), ribonuclease P protein component (14.96 kDa) and cobalt-precorrin-5B C(1)-methyltransferase (38.43 kDa) with MASCOT score of 54, 81, 94, and 100, respectively. All of these proteins help the bacteria to overcome arsenate stress.

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes.

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.