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Due to the rise of multidrug-resistant strains of Vibrio cholerae and the recent cholera outbreaks in African and Asian nations, it is imperative to identify novel therapeutic targets and possible vaccine candidates. In this regard, this work primarily aims to identify and characterize new antigenic molecules using comparative RNA sequencing data and label-free proteomics data, carried out with essential GTPase cgtA knockdown and wild-type strain of V. cholerae. We identified hitherto 51 characterized proteins from high-throughput RNA-sequencing and proteomics data. This work involved the assessment of their physicochemical characteristics, subcellular localization, solubility, structures, and functional annotations. In addition, the immunoinformatic and reverse vaccinology technique was used to find new vaccine targets with high antigenicity, low allergenicity, and low toxicity profiles. Among the 51 proteins, 24 were selected based on their immunogenic profiles to identify B/T-cell epitopes. In addition, 20 prospective therapeutic targets were identified using virulence predictions and related investigations. Furthermore, two proteins, UniProt ID- Q9KRD2 and Q9KU58, with molecular weight of 92kDa and 12kDa, respectively, were chosen for cloning and expression towards in vitro biochemical characterization based on their range of expression patterns, high antigenic, low allergenic, and low toxicity properties. In conclusion, we believe that this study will reveal new facets and avenues for drug discovery and put us a step forward toward novel therapeutic interventions against the deadly disease of cholera., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Basu Mallick et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CgtA is an essential bacterial GTPase consisting of a highly conserved N-terminal Spo 0B -associated G TP-binding protein (Obg) domain, a central GTPase domain, and a variable C-terminal domain (CTD). This study reports global changes in the proteome and transcriptome of wild-type (Wt) versus full-length CgtA-depleted Vibrio cholerae in minimal media. Comparative transcriptome sequencing (RNA-Seq), followed by comparative proteomic analyses, revealed that the knockdown of cgtA significantly altered expressions of 311 proteins involved in diverse cellular activities, many of which are associated with the survival of V. cholerae. Various intracellular functional roles of CgtA in growth, viability, motility, morphology, and persister phenotypes in V. cholerae are revealed based on subsequent confirmatory experiments. Furthermore, a more sustained mRNA expression pattern of cgtA in a minimal medium than in a rich medium was also observed for Wt V. cholerae, where the highest level of mRNA expression of cgtA was observed during the logarithmic growth phase. Thereby, we propose that minimal medium-associated reduced growth rate coupled with cgtA depletion aggravates the intracellular stress in V. cholerae, interrupting vital cellular processes. The functional role of the CTD in V. cholerae is not fully understood. Hence, to specifically investigate the intracellular role of the 57-amino-acid-long CTD of CgtA VC , the CTD-only portion of CgtA was deleted. Subsequent proteomics studies revealed an altered expression of 240 proteins in the CgtA(ΔCTD) mutant, having major overlap with the full-length cgtA -deleted condition. Overall, our study reveals an alternative facet of the survival mechanism of V. cholerae during nutritional downshift as per the concomitant consequences of cgtA depletion. IMPORTANCE It is very important that we must find new drug target proteins from multidrug-resistant human-pathogenic organisms like V. cholerae. CgtA is among such potential candidates, and here, we are reporting about some newly identified cellular roles of it that are important for the survival of V. cholerae. Briefly, we knocked down the full-length cgtA gene, as well as did a partial deletion of this gene from the V. cholerae genome followed by RNA-Seq and proteomics studies. Results from our study revealed up- and downregulation of several known and unknown genes and proteins as the effect of the cgtA knockdown experiment. Also, we have presented some interesting observations that are linked with cgtA for growth, viability, motility, morphology, and persister phenotypes in V. cholerae. Our study enhances the importance of CgtA and paves the way for further exploration based on our provided data.

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The present study characterized aresenate reductase of Bacillus thuringiensis KPWP1, tolerant to salt, arsenate and a wide range of pH during growth. Interestingly, it was found that arsC, arsB and arsR genes involved in arsenate tolerance are distributed in the genome of strain KPWP1. The inducible arsC gene was cloned, expressed and the purified ArsC protein showed profound enzyme activity with the K M and K cat values as 25 µM and 0.00119 s -1 , respectively. In silico studies revealed that in spite of 19-26% differences in gene sequences, the ArsC proteins of Bacillus thuringiensis, Bacillus subtilis and Bacillus cereus are structurally conserved and ArsC structure of strain KPWP1 is close to nature. Docking and analysis of the binding site showed that arsenate ion interacts with three cysteine residues of ArsC and predicts that the ArsC from B. thuringiensis KPWP1 reduces arsenate by using the triple Cys redox relay mechanism., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Protein 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., Results: Our 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., Conclusions: From 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.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1 mt ) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1 mt in mitochondrial tRNA (tRNA mt ) translocation. In particular, the mito-specific C-terminal extension in EF-G1 mt is directly involved in translocation of the acceptor arm of the A-site tRNA mt . In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

CgtA is an essential bacterial GTPase protein involved in multiple cellular activities. In the presence of 50S ribosome, its GTPase activity increases significantly. Through sequential deletions of CgtA protein of Vibrio cholerae (CgtA vc ) we found that its N terminal Obg domain is essential for ribosome binding and augmenting the ribosome mediated GTPase activity. Strategic deletions of the three glycine rich loops of Obg domain revealed that loop 1 of Obg domain is involved in anchoring the protein into the 50S, whereas, loop 2 & loop 3 are involved in conveying the effect of interaction of the Obg domain with the 50S to the GTPase domain through an interdomain linker, followed by GTP hydrolysis. On the other hand, the non-conserved C-terminal domain (CTD) is not directly involved in ribosome binding but shows negative impact on GTPase activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CgtA is an essential ribosome associated GTPase protein of bacteria. It has three domains, viz., Obg, GTPase, and C-terminal domain. It is a multifunctional protein and it is being considered as a potential drug target against bacterial infections. Despite the importance, CgtA's action mechanisms are not well known which warrants further study. Towards that goal, we are pursuing biochemical and computational studies in Vibrio cholerae CgtA (CgtA vc ). Biochemically we found that a single amino acid substitution from Gly98 to Asp98 belonging to the Obg domain caused reduced GTPase activity of CgtA vc . The results from our comparative MD simulations studies revealed that in silico amino acid substitution for Gly98Asp influenced the inter-domain movement between Obg domain and GTPase domain. Moreover, we found significant alteration of intra-domain movements among the P-loop, G4 box, and G5 box of the GTPase domain, implying a potential cause for the reduced GTPase activity.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

A new class of H-bond donating ureas was developed for the ring-opening polymerization (ROP) of lactone monomers, and they exhibit dramatic rate acceleration versus previous H-bond mediated polymerization catalysts. The most active of these new catalysts, a tris-urea H-bond donor, is among the most active organocatalysts known for ROP, yet it retains the high selectivity of H-bond mediated organocatalysts. The urea cocatalyst, along with an H-bond accepting base, exhibits the characteristics of a "living" ROP, is highly active, in one case, accelerating a reaction from days to minutes, and remains active at low catalyst loadings. The rate acceleration exhibited by this H-bond donor occurs for all base cocatalysts examined. A mechanism of action is proposed, and the new catalysts are shown to accelerate small molecule transesterifications versus currently known monothiourea catalysts. It is no longer necessary to choose between a highly active or highly selective organocatalyst for ROP.

For the first time, the controlled ring-opening polymerization (ROP) of ε-thionocaprolactone (tnCL) is conducted. The organocatalytic ROP of tnCL occurs without carbonyl scrambling, leading to homopoly(ε-thionocaprolactone) (PtnCL). The ROP by base catalysts alone is proposed to proceed via a nucleophilic mechanism, while the addition of an H-bond donating thiourea (TU) is shown to provide excellent reaction control. The increased reaction control provided by the TU occurs in the virtual absence of binding between tnCL and TU, and a mechanistic account for this observation is discussed. The monomer ring strain is measured and found to be similar to δ-valerolactone (VL). Copolymers with VL are synthesized, and the resulting analysis of the copolymer materials properties provides the only known physical characterizations of poly(thio(no)ester- co -ester)s.

CgtA is a highly conserved ribosome binding protein involved in ribosome biogenesis and associated with stringent response. It is a 55 KDa GTPase protein consisting of GTPase, Obg and C-terminal domains. The function of the latter two domains was not clear and despite the importance, the mode of action of CgtA is still largely unknown. Knocking out of CgtA gene is lethal and mutations lead to growth, sporulation and developmental defects in bacteria. It was found that a growth defect and pinhole size colony morphology of Bacillus subtilis was associated with a Gly92Asp point mutation on the Obg domain of its CgtA protein, instead of its GTPase domain. CgtA is an important and essential protein of the deadly diarrhea causing bacteria Vibrio cholerae and in order to investigate the mode of action of the V. cholerae CgtA we have utilized this information. We measured the GTPase activity of V. cholerae CgtA (CgtAvc) protein in the presence of purified ribosome. Our results showed 5-fold increased GTP hydrolysis activity compared to its intrinsic activity. Then we explored the GTPase activity of the mutated CgtAvc (Gly98Asp) located at the Obg domain, which reduced the GTP hydrolysis rate to half. The double point mutations (Gly98Asp, and Tyr194Gly) encompassing another conserved residue, Tyr194, located at the diagonally opposite position in the GTPase domain largely restored (about 82%) the reduced GTPase activity, revealing a fine-tuned inter-domain movement readily associated with the GTPase activity of CgtA and thus maintaining the proper functioning of the CgtA protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Thiourea (TU)/amine base cocatalysts are commonly employed for well-controlled, highly active "living" organocatalytic ring-opening polymerizations (ROPs) of cyclic esters and carbonates. In this work, several of the most active cocatalyst pairs are shown by 1 H NMR binding studies to be highly associated in solution, dominating all other known noncovalent catalyst/reagent interactions during ROP. One strongly binding catalyst pair behaves kinetically as a unimolecular catalyst species. The high selectivity and activity exhibited by these ROP organocatalysts are attributed to the strong binding between the two cocatalysts, and the predictive utility of these binding parameters is applied for the discovery of a new, highly active cocatalyst pair.

Ecliptaalba (Bhringraj) is an important traditional Indian medicinal plant reported to have antipyretic, anti-inflammatory, antihistaminic, hepatoprotective and anthelmintic effect well documented in literature. In the present study ethanolic leaf extract of eclipta alba (EEEA) was evaluated for its haemostatic activity by their ability to affect the screening test or modify the experimentally induced prolongation of test time on the basis of its folklore claim in normal, aspirin and heparin treated rabbits. The parameters recorded were BT, CT, PT and APTT & platelet count in all the three groups. The study was carried out in 3 parts in different doses 200,400,600 & 800mg/kg. Part-A-study of the haemostatic effect of Ethanolic extract of E. alba (EEEA) in graded doses on normal rabbits. Part-B-study of EEEA in aspirin treated rabbits. Part-C-study of EEEA in heparin treated rabbits. Tranexamic acid was used as the standard drug. Statistical analysis was performed using one way ANOVA & student t test. In normal rabbits EEEA high doses 800mg/k & 600mg/kg exhibited significant reduction in BT & CT (p<0.01). The same doses were found to decrease the aspirin induced increase in BT & heparin induced increase in CT in a significant manner (p<0.01). As decrease in CT might be due to liberation of some active principles from leaves of Ecliptaalba which demonstrated to have anti-haemorrhagic activity against snake bite not affecting any coagulation factor. No significant effect on PT, APPT and platelet count was observed. EEEA exhibited definite haemostatic effect in the above 3 sets of study. Phytochemical analysis and isolation of active principle is essential to carry on beneficial effects from laboratory bench to bedside of patients.

Pharmacology is a subject taught in the medical curriculum in India over a period of one and half years along with pathology, microbiology and forensic medicine. The present study was planned to know the opinion of medical students regarding pharmacology and to assess the proposed teaching schedule and methods of teaching pharmacology. The study was conducted in a private medical college in eastern India among the medical undergraduate students in 5th semester. Total 74 students participated in the study. A pre-designed, pre-tested, semi-structured questionnaire was given to the students and data was collected after one hour. Collected data was compiled, tabulated and analyzed in SPSS (version 16.0). The subject was perceived as interesting and useful by majority of students and most of them were in opinion to integrate pharmacology with the clinical subjects. Lecture in whole class was the most preferred teaching method according to the students and teaching with chalk and board they preferred most. Rational use of medicine, clinical trial, pediatric and geriatric pharmacology are the important topics the students felt to be included in the curriculum. Regular assessment of teaching methods by the students and taking suggestions from the students about improving the teaching method and redesigning the curriculum can help a lot in improving the learning capacity of the medical students and that will give benefit for the society as a whole.

Aims: Cervical spinal cord injury is a major cause of fatality among trauma victims. Unlike developed countries, national spinal cord injury database is not maintained in India. Paucity of data hampers the understanding of epidemiology of the event in India. So, this study was conducted to ascertain the epidemiological pattern of death due to cervical spinal cord injury and to identify the risk factors; also to find the association of vertebral injuries with various epidemiological parameters., Methods: Among autopsies conducted in Burdwan Medical College and Hospital from 2000 to 2010, cases with autopsy report mentioning "death due to cervical spinal cord injury" either primary or secondary were included in the study. It was a retrospective observational study based on autopsy report., Results: 536 cases met the inclusion criteria. Among them 89.4% were male and 10.6% were female; 63.8% victims were young adults (20-39 years). Mean (plus or minus S.D.) age was 33.5 years (plus or minus 12.8). All, apart from 5.4%, suffered from single segment injury with commonest site being C3-C4 (37.3%). Highest number of trauma occurred between 6 am to 9 am, 56.6% of the victims died before hospitalisation. Commonest mode of injury was road traffic accident (52.2%) followed by fall from height (25.0%); 53.7% cases had vertebral fracture with dislocation, 34.3% had only vertebral fracture. Only dislocation was significantly high up to 19 years age (p<0.001) and among females (p=0.029). All elderly suffered from fracture., Conclusions: This study revealed an unusually high male:female ratio with much representation of the younger age groups in the injuries in comparison to other parts of world. In absence of national database, this result can be used as a surrogate data.

Mitochondria have their own translational machineries for the synthesis of thirteen polypeptide chains that are components of the complexes that participate in the process of oxidative phosphorylation (or ATP generation). Translation initiation in mammalian mitochondria requires two initiation factors, IF2(mt) and IF3(mt), instead of the three that are present in eubacteria. The mammalian IF2(mt) possesses a unique 37 amino acid insertion domain, which is known to be important for the formation of the translation initiation complex. We have obtained a three-dimensional cryoelectron microscopic map of the mammalian IF2(mt) in complex with initiator fMet-tRNA(iMet) and the eubacterial ribosome. We find that the 37 amino acid insertion domain interacts with the same binding site on the ribosome that would be occupied by the eubacterial initiation factor IF1, which is absent in mitochondria. Our finding suggests that the insertion domain of IF2(mt) mimics the function of eubacterial IF1, by blocking the ribosomal aminoacyl-tRNA binding site (A site) at the initiation step.

Plastid-specific ribosomal proteins (PSRPs) have been proposed to play roles in the light-dependent regulation of chloroplast translation. Here we demonstrate that PSRP1 is not a bona fide ribosomal protein, but rather a functional homologue of the Escherichia coli cold-shock protein pY. Three-dimensional Cryo-electron microscopic (Cryo-EM) reconstructions reveal that, like pY, PSRP1 binds within the intersubunit space of the 70S ribosome, at a site overlapping the positions of mRNA and A- and P-site tRNAs. PSRP1 induces conformational changes within ribosomal components that comprise several intersubunit bridges, including bridge B2a, thereby stabilizes the ribosome against dissociation. We find that the presence of PSRP1/pY lowers the binding of tRNA to the ribosome. Furthermore, similarly to tRNAs, PSRP1/pY is recycled from the ribosome by the concerted action of the ribosome-recycling factor (RRF) and elongation factor G (EF-G). These results suggest a novel function for EF-G and RRF in the post-stress return of PSRP1/pY-inactivated ribosomes to the actively translating pool.

The mechanism of translation in eubacteria and organelles is thought to be similar. In eubacteria, the three initiation factors IF1, IF2, and IF3 are vital. Although the homologs of IF2 and IF3 are found in mammalian mitochondria, an IF1 homolog has never been detected. Here, we show that bovine mitochondrial IF2 (IF2(mt)) complements E. coli containing a deletion of the IF2 gene (E. coli DeltainfB). We find that IF1 is no longer essential in an IF2(mt)-supported E. coli DeltainfB strain. Furthermore, biochemical and molecular modeling data show that a conserved insertion of 37 amino acids in the IF2(mt) substitutes for the function of IF1. Deletion of this insertion from IF2(mt) supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. These observations provide strong evidence that a single factor (IF2(mt)) in mammalian mitochondria performs the functions of two eubacterial factors, IF1 and IF2.

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes. Fitting of the conserved portions of the x-ray crystallographic structure of the bacterial 70S ribosome into our cryo-EM map of the chloro-ribosome reveals the positions of PRP extensions and the locations of the PSRPs. Surprisingly, PSRP1 binds in the decoding region of the small (30S) ribosomal subunit, in a manner that would preclude the binding of messenger and transfer RNAs to the ribosome, suggesting that PSRP1 is a translation factor rather than a ribosomal protein. PSRP2 and PSRP3 appear to structurally compensate for missing segments of the 16S rRNA within the 30S subunit, whereas PSRP4 occupies a position buried within the head of the 30S subunit. One of the two PSRPs in the large (50S) ribosomal subunit lies near the tRNA exit site. Furthermore, we find a mass of density corresponding to chloro-ribosome recycling factor; domain II of this factor appears to interact with the flexible C-terminal domain of PSRP1. Our study provides evolutionary insights into the structural and functional roles that the PSRPs play during protein synthesis in chloroplasts.

Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.

After the termination step of translation, the posttermination complex (PoTC), composed of the ribosome, mRNA, and a deacylated tRNA, is processed by the concerted action of the ribosome-recycling factor (RRF), elongation factor G (EF-G), and GTP to prepare the ribosome for a fresh round of protein synthesis. However, the sequential steps of dissociation of the ribosomal subunits, and release of mRNA and deacylated tRNA from the PoTC, are unclear. Using three-dimensional cryo-electron microscopy, in conjunction with undecagold-labeled RRF, we show that RRF is capable of spontaneously moving from its initial binding site on the 70S Escherichia coli ribosome to a site exclusively on the large 50S ribosomal subunit. This movement leads to disruption of crucial intersubunit bridges and thereby to the dissociation of the two ribosomal subunits, the central event in ribosome recycling. Results of this study allow us to propose a model of ribosome recycling.

During tRNA translocation on the ribosome, an arc-like connection (ALC) is formed between the G' domain of elongation factor G (EF-G) and the L7/L12-stalk base of the large ribosomal subunit in the GDP state. To delineate the boundary of EF-G within the ALC, we tagged an amino acid residue near the tip of the G' domain of EF-G with undecagold, which was then visualized with three-dimensional cryo-electron microscopy (cryo-EM). Two distinct positions for the undecagold, observed in the GTP-state and GDP-state cryo-EM maps of the ribosome bound EF-G, allowed us to determine the movement of the labeled amino acid. Molecular analyses of the cryo-EM maps show: (1) that three structural components, the N-terminal domain of ribosomal protein L11, the C-terminal domain of ribosomal protein L7/L12, and the G' domain of EF-G, participate in formation of the ALC; and (2) that both EF-G and the ribosomal protein L7/L12 undergo large conformational changes to form the ALC.

Linezolid, an oxazolidinone antibiotic, inhibits bacterial protein synthesis by binding to 23S ribosomal RNA (rRNA). We studied 3 patients who experienced lactic acidosis while receiving linezolid therapy. The toxicity may have been caused by linezolid binding to mitochondrial 16S rRNA. Genetic polymorphisms may have contributed to the toxicity in 2 patients.

When exponentially growing Vibrio cholerae cells were shifted from 37 degrees C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15 degrees C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA(VC)) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspA(VC) and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspA(VC) and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37 degrees C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.

The mitochondrial ribosome is responsible for the biosynthesis of protein components crucial to the generation of ATP in the eukaryotic cell. Because the protein:RNA ratio in the mitochondrial ribosome (approximately 69:approximately 31) is the inverse of that of its prokaryotic counterpart (approximately 33:approximately 67), it was thought that the additional and/or larger proteins of the mitochondrial ribosome must compensate for the shortened rRNAs. Here, we present a three-dimensional cryo-electron microscopic map of the mammalian mitochondrial 55S ribosome carrying a tRNA at its P site, and we find that instead, many of the proteins occupy new positions in the ribosome. Furthermore, unlike cytoplasmic ribosomes, the mitochondrial ribosome possesses intersubunit bridges composed largely of proteins; it has a gatelike structure at its mRNA entrance, perhaps involved in recruiting unique mitochondrial mRNAs; and it has a polypeptide exit tunnel that allows access to the solvent before the exit site, suggesting a unique nascent-polypeptide exit mechanism.