RESUMO
The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.
Assuntos
Microscopia Crioeletrônica , Ciclopentanos , Ácido Fusídico , Fator G para Elongação de Peptídeos , Ribossomos , Staphylococcus aureus , Ácido Fusídico/farmacologia , Ácido Fusídico/química , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Ciclopentanos/farmacologia , Ciclopentanos/química , Fator G para Elongação de Peptídeos/metabolismo , Fator G para Elongação de Peptídeos/química , Antibacterianos/farmacologia , Antibacterianos/química , Modelos Moleculares , RNA de Transferência/metabolismo , RNA de Transferência/químicaRESUMO
During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Listeria monocytogenes , Ribossomos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Ribossomos/metabolismo , Listeria monocytogenes/metabolismo , Listeria monocytogenes/genética , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/química , Biossíntese de Proteínas , Modelos Moleculares , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Fator G para Elongação de Peptídeos/metabolismo , Fator G para Elongação de Peptídeos/químicaRESUMO
The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit rotates counterclockwise relative to the large subunit, placing the ribosome into the rotated conformation. Simultaneously, tRNAs move into the hybrid conformation, and the L1 stalk moves inward toward the P-site tRNA. The conformational dynamics of pretranslocation ribosomes were extensively studied by ensemble and single-molecule methods. Different experimental modalities tracking ribosomal subunits, tRNAs, and the L1 stalk showed that pretranslocation ribosomes undergo spontaneous conformational transitions. Thus, peptidyl transfer unlocks the ribosome and decreases an energy barrier for the reverse ribosome rotation during translocation. However, the tracking of translation with ribosomes labeled at rRNA helices h44 and H101 showed a lack of spontaneous rotations in pretranslocation complexes. Therefore, reverse intersubunit rotations occur during EF-G catalyzed translocation. To reconcile these views, we used high-speed single-molecule microscopy to follow translation in real time. We showed spontaneous rotations in puromycin-released h44-H101 dye-labeled ribosomes. During elongation, the h44-H101 ribosomes undergo partial spontaneous rotations. Spontaneous rotations in h44-H101-labeled ribosomes are restricted prior to aminoacyl-tRNA binding. The pretranslocation h44-H101 ribosomes spontaneously exchanged between three different rotational states. This demonstrates that peptidyl transfer unlocks spontaneous rotations and pretranslocation ribosomes can adopt several thermally accessible conformations, thus supporting the Brownian model of translocation.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Ribossomos , Ribossomos/metabolismo , RNA de Transferência/metabolismo , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de ProteínasRESUMO
In vivo protein synthesis is crucial for all domains of life. It is accomplished through translational machinery, and a key step is the translocation of tRNA-mRNA by elongation factor G (EF-G). Genome-based analysis revealed two EF-G encoding genes (S0885 and S2082) in the freshwater cyanobacterium model Synechococcus elongatus PCC7942. S0885 is the essential EF-G gene for photosynthesis. We generated a strain of S. elongatus PCC7942 that overexpressed S0885 (OX-S0885) to identify EF-G functionality. RT-PCR and Western blot analyses revealed increased transcriptional and translational levels in OX-S0885 at 10.5-13.5 and 2.0-3.0 fold, respectively. Overexpression of S0885 led to an increase in specific growth rate. Additionally, polysome-to-monosome ratio (P/M) and RNA-to-protein ratio (R/P) were elevated in OX-S0885 compared with the empty vector. Interestingly, R/P in OX-S0885 was retained at more than 70% under oxidative stress while R/P in the empty vector was severely depleted, suggesting the maintenance of translation. Thus, S0885 appeared to be the important target of oxidative stress because it was protected by the stress response system to maintain its function. These results suggest that cyanobacterial EF-G has a primary function in translation and an unrelated activity during stress conditions. These findings support the substantial role of EF-G in the formation and maintenance of cellular protein formation, and in the protection of the global translational mechanism under oxidative stress condition.
Assuntos
Fator G para Elongação de Peptídeos , Synechococcus , Synechococcus/genética , Western Blotting , Biossíntese de ProteínasRESUMO
Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Fator G para Elongação de Peptídeos , Ribossomos , Guanosina Trifosfato/metabolismo , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Ribossomos/química , Ribossomos/metabolismo , RNA Ribossômico 23S/metabolismo , RNA de Transferência/metabolismoRESUMO
Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.
Assuntos
Enterococcus faecalis , Fator G para Elongação de Peptídeos , Humanos , Fator G para Elongação de Peptídeos/metabolismo , Fator G para Elongação de Peptídeos/farmacologia , Antibacterianos/farmacologia , Enterococcus/metabolismo , Biofilmes , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatos/metabolismoRESUMO
OBJECTIVES: Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. METHODS: Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MICâ=â4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. RESULTS: WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520Iâ+âR635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. CONCLUSIONS: We describe the first high-level gentamicin-resistant gonococcal isolate (MICâ=â128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Gentamicinas/farmacologia , Fator G para Elongação de Peptídeos , Gonorreia/tratamento farmacológico , Testes de Sensibilidade Microbiana , Farmacorresistência BacterianaRESUMO
In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection.
Assuntos
Fator G para Elongação de Peptídeos , Ribossomos , RNA Mensageiro/metabolismo , Fator G para Elongação de Peptídeos/genética , Ribossomos/genética , Ribossomos/metabolismo , Biossíntese de Proteínas/genética , Códon/análise , Códon/metabolismo , Fases de Leitura , RNA de Transferência/genéticaRESUMO
Translational G proteins, whose release from the ribosome is triggered by GTP hydrolysis, regulate protein synthesis. Concomitantly with binding and dissociation of protein factors, translation is accompanied by forward and reverse rotation between ribosomal subunits. Using single-molecule measurements, we explore the ways in which the binding of translational GTPases affects inter-subunit rotation of the ribosome. We demonstrate that the highly conserved translation factor LepA, whose function remains debated, shifts the equilibrium toward the non-rotated conformation of the ribosome. By contrast, the catalyst of ribosome translocation, elongation factor G (EF-G), favors the rotated conformation of the ribosome. Nevertheless, the presence of P-site peptidyl-tRNA and antibiotics, which stabilize the non-rotated conformation of the ribosome, only moderately reduces EF-G binding. These results support the model suggesting that EF-G interacts with both the non-rotated and rotated conformations of the ribosome during mRNA translocation. Our results provide new insights into the molecular mechanisms of LepA and EF-G action and underscore the role of ribosome structural dynamics in translation.
Assuntos
GTP Fosfo-Hidrolases , Biossíntese de Proteínas , Humanos , GTP Fosfo-Hidrolases/genética , Fator G para Elongação de Peptídeos/metabolismo , Rotação , Ribossomos/metabolismo , Translocação Genética , RNA de Transferência/genéticaRESUMO
In translation initiation in prokaryotes, IF3 recognizes the interaction between the initiator codon of mRNA and the anticodon of fMet-tRNAini and then relocates the fMet-tRNAini to an active position. Here, we have surveyed 328 codon-anticodon combinations for the preference of IF3. At the first and second base of the codon, only Watson-Crick base pairs are tolerated. At the third base, stronger base pairs, for example, Watson-Crick, are more preferred, but other types of base pairs, for example, G/U wobble, are also tolerated; weaker base pairs are excluded by IF3. When the codon-anticodon combinations are unfavorable for IF3 or the concentration of IF3 is too low to recognize any codon-anticodon combinations, IF3 fails to set the P-site fMet-tRNAini at the active position and causes its drop-off from the ribosome. Thereby, translation reinitiation occurs from the second aminoacyl-tRNA at the A site to yield a truncated peptide lacking the amino-terminal fMet. We refer to this event as the amino-terminal drop-off-reinitiation. We also showed that EF-G and RRF are involved in disassembling such an aberrant ribosome complex bearing inactive fMet-tRNAini Thereby EF-G and RRF are able to exclude unfavorable codon-anticodon combinations with weaker base pairs and alleviate the amino-terminal drop-off-reinitiation.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos , Anticódon/genética , Códon/genética , Escherichia coli/genética , Fator G para Elongação de Peptídeos/genética , Peptídeos , RNA de Transferência/genética , Perforina/metabolismoRESUMO
Protein synthesis is crucial for cell growth and survival yet one of the most energy-consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA-tRNAs through the ribosome, bacterial elongation factor G (EF-G) hydrolyzes energy-rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF-G paralog-EF-G2-that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF-G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF-G2 is ~10-fold more abundant than canonical EF-G1 in bacteria harvested from murine ceca and, unlike EF-G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26-residue region unique to EF-G2 that is essential for protein synthesis, EF-G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient-fluctuating environments.
Assuntos
Bacteroides , Fator G para Elongação de Peptídeos , Animais , Camundongos , Bacteroides/genética , Bacteroides/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/química , Ribossomos/metabolismo , RNA de Transferência/metabolismoRESUMO
BACKGROUND: Combined oxidative phosphorylation deficiency-1 (COXPD1) resulting from a mutation in the G elongation factor mitochondrial 1 (GFM1) gene is an autosomal recessive multisystem disorder arising from a defect in the mitochondrial oxidative phosphorylation system. Death usually appears in the first weeks or years of lifespan. CASE: We report a male patient with ventriculomegaly diagnosed in the 8th month of pregnancy. The delivery was done by caesarean section and respiratory failure occurred immediately after birth. Hypoglycemia, lactic acidosis, elevated gamma-glutamyl transferase and hepatomegaly were confirmed. The brain MRI detected hypoplasia of the cerebellar hemispheres, dilated lateral ventricles, and markedly immature brain parenchyma. Epilepsy had been present since the third month. At 5 months of age, neurological follow-up showed his head circumference to be 37 cm, with plagiocephaly, a low hairline, a short neck, axial hypotonia and he did not adopt any developmental milestones. A genetic mutation, a missense variant in the GFM1 gene, was confirmed: c.748C > T (p.Arg250Trp) was homozygous in the GFM1 gene. CONCLUSIONS: To the best of our knowledge, 28 cases of COXPD1 disease caused by mutations in the GFM1 gene have been described in the literature. COXPD1 should be considered due to symptoms and signs which begin during intrauterine life or at birth. Signs of impaired energy metabolism should indicate that the disease is in the group of metabolic encephalopathies.
Assuntos
Encefalopatia Hepática , Erros Inatos do Metabolismo , Doenças Mitocondriais , Feminino , Humanos , Recém-Nascido , Masculino , Gravidez , Cesárea , Proteínas Mitocondriais , Mutação , Fator G para Elongação de Peptídeos , SérviaRESUMO
Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.
Assuntos
Fator G para Elongação de Peptídeos , Ribossomos , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/química , Guanosina Trifosfato/química , Hidrólise , Ribossomos/metabolismo , RNA de Transferência/química , RNA Mensageiro/química , GTP Fosfo-Hidrolases/genética , Pirenos/análise , GuanosinaRESUMO
The ribosome is a complex biomolecular machine that utilizes large-scale conformational rearrangements to synthesize proteins. For example, during the elongation cycle, the "head" domain of the ribosomal small subunit (SSU) is known to undergo transient rotation events that allow for movement of tRNA molecules (i.e., translocation). While the head may exhibit rigid-body-like properties, the precise relationship between experimentally accessible probes and multidimensional rotations has yet to be established. To address this gap, we perform molecular dynamics simulations of the translocation step of the elongation cycle in the ribosome, where the SSU head spontaneously undergoes rotation and tilt-like motions. With this data set (1250 simulated events), we used statistical and information-theory-based measures to identify possible single-molecule probes that can isolate SSU head rotation and head tilting. This analysis provides a molecular interpretation for previous single-molecule measurements, while establishing a framework for the design of next-generation experiments that may precisely probe the mechanistic and kinetic aspects of the ribosome.
Assuntos
RNA de Transferência , Ribossomos , Ribossomos/metabolismo , RNA de Transferência/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Cinética , Biossíntese de Proteínas , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismoRESUMO
Ribosomes are at the core of the central dogma of life. They perform the last major step of gene expression by translating the information written in the nucleotide codon sequences into the amino acid sequence of a protein. This is a complex mechanochemical process that requires the coordination of multiple dynamic events within the ribosome such as the precise timing of decoding and the subsequent translocation along the mRNA. We have previously used a high-resolution optical tweezers instrument with single-molecule fluorescence capabilities ("fleezers") to study how ribosomes couple binding of the GTPase translation elongation factor EF-G with internal conformational changes to unwind and progress across the mechanical barriers posed by mRNA secondary structures. Here, we present a detailed description of the procedures for monitoring two orthogonal channels (EF-G binding and translocation) by single actively translating ribosomes in real-time, to uncover the mechanism by which they harness chemical energy to generate mechanical force and displacement.
Assuntos
Escherichia coli , Fator G para Elongação de Peptídeos , Escherichia coli/genética , Fator G para Elongação de Peptídeos/análise , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
Translocation of messenger RNA (mRNA) and transfer RNA (tRNA) substrates through the ribosome during protein synthesis, an exemplar of directional molecular movement in biology, entails a complex interplay of conformational, compositional, and chemical changes. The molecular determinants of early translocation steps have been investigated rigorously. However, the elements enabling the ribosome to complete translocation and reset for subsequent protein synthesis reactions remain poorly understood. Here, we have combined molecular simulations with single-molecule fluorescence resonance energy transfer imaging to gain insights into the rate-limiting events of the translocation mechanism. We find that diffusive motions of the ribosomal small subunit head domain to hyper-swivelled positions, governed by universally conserved rRNA, can maneuver the mRNA and tRNAs to their fully translocated positions. Subsequent engagement of peptidyl-tRNA and disengagement of deacyl-tRNA from mRNA, within their respective small subunit binding sites, facilitate the ribosome resetting mechanism after translocation has occurred to enable protein synthesis to resume.
Assuntos
Fator G para Elongação de Peptídeos , Ribossomos , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , RNA de Transferência/metabolismo , Ribossomos/metabolismoRESUMO
Mitochondrial protein synthesis requires three elongation factors including EF-Tu (TUFM; OMIM 602389), EF-Ts (TSFM; OMIM 604723), and EF-G1 (GFM1; OMIM 606639). Pathogenic variants in any of these three members result in defective mitochondrial translation which can impart an oxidative phosphorylation (OXPHOS) deficiency. In this study, we investigated a consanguineous Pakhtun Pakistani family. There were four affected siblings at the time of this study and one affected girl had died in infancy. The index patient had severe intellectual disability, global developmental delay, dystonia, no speech development, feeding difficulties, and nystagmus. MRI brain presented thinning of corpus callosum and polymicrogyria. Whole exome sequencing revealed a novel compound heterozygous variant in GFM1 located on chromosome 3q25.32. Sanger sequencing confirmed recessive segregation of the maternal (NM_001308164.1:c.409G > A; p.Val137Met) and paternal (NM_001308164.1:c.1880G > A; p.Arg627Gln) variants in all the four affected siblings. These variants are classified as "likely-pathogenic" according to the recommendation of ACMG/AMP guideline. GFM1 alterations mostly lead to severe phenotypes and the patients may die in early neonatal life; however, four of the affected siblings had survived till the ages of 10-17 years, without developing any life-threatening conditions. Mostly, in cousin marriages, the pathogenic variants are identical-by-descent, and affected siblings born to such parents are homozygous. Three homozygous variants were shortlisted in the analysis of the WES data, but Sanger sequencing did not confirm their segregation with the disease phenotype. This is the first report from Pakistan expanding pathogenicity of GFM1 gene.
Assuntos
Distonia , Distúrbios Distônicos , Deficiência Intelectual , Polimicrogiria , Distonia/genética , Exoma/genética , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Proteínas Mitocondriais/genética , Mutação , Linhagem , Fator G para Elongação de Peptídeos/genética , Fatores de Alongamento de Peptídeos/genética , Polimicrogiria/genética , Sequenciamento do ExomaRESUMO
Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.
Assuntos
Antibacterianos , Fator G para Elongação de Peptídeos , Antibacterianos/metabolismo , Ácido Fusídico/farmacologia , Humanos , Oligopeptídeos , Fator G para Elongação de Peptídeos/metabolismo , Ribossomos/metabolismo , Translocação GenéticaRESUMO
The continued emergence of Neisseria gonorrhoeae isolates which are resistant to first-line antibiotics has reinvigorated interest in alternative therapies such as expanded use of gentamicin (Gen). We hypothesized that expanded use of Gen promotes emergence of gonococci with clinical resistance to this aminoglycoside. To understand how decreased susceptibility of gonococci to Gen might develop, we selected spontaneous low-level Gen-resistant (GenR) mutants (Gen MIC = 32 µg/mL) of the Gen-susceptible strain FA19. Consequently, we identified a novel missense mutation in fusA, which encodes elongation factor G (EF-G), causing an alanine (A) to valine (V) substitution at amino acid position 563 in domain IV of EF-G; the mutant allele was termed fusA2. Transformation analysis showed that fusA2 could increase the Gen MIC by 4-fold. While possession of fusA2 did not impair either in vitro gonococcal growth or protein synthesis, it did result in a fitness defect during experimental infection of the lower genital tract in female mice. Through bioinformatic analysis of whole-genome sequences of 10,634 international gonococcal clinical isolates, other fusA alleles were frequently detected, but genetic studies revealed that they could not decrease Gen susceptibility in a similar manner to fusA2. In contrast to these diverse international fusA alleles, the fusA2-encoded A563V substitution was detected in only a single gonococcal clinical isolate. We hypothesize that the rare occurrence of fusA2 in N. gonorrhoeae clinical isolates is likely due to a fitness cost during infection, but compensatory mutations which alleviate this fitness cost could emerge and promote GenR in global strains.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Substituição de Aminoácidos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana/genética , Feminino , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Gonorreia/tratamento farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Fator G para Elongação de PeptídeosRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) can cause systemic infections in both humans and animals. As an essential nutrient, iron is strictly sequestered by the host. Circumventing iron sequestration is a determinant factor for ExPEC infection. However, the ExPEC iron acquisition mechanism, particularly the mechanism of transferrin (TF) acquisition, remains unclear. This study reports that iron-saturated holo-TF can be utilized by ExPEC to promote its growth in culture medium and survival in macrophages. ExPEC specifically bound to holo-TF instead of iron-free apo-TF via the surface located elongation factor G (EFG) in both culture medium and macrophages. As a moonlighting protein, EFG specifically bound holo-TF and also released iron in TF. These two functions were performed by different domains of EFG, in which the N-terminal domains were responsible for holo-TF binding and the C-terminal domains were responsible for iron release. The functions of EFG and its domains have also been further confirmed by surface-display vectors. The surface overexpression of EFG bound significantly more holo-TF in macrophages and significantly improved bacterial intracellular survival ability. Our findings reveal a novel iron acquisition mechanism involving EFG, which suggests novel research avenues into the molecular mechanism of ExPEC resistance to nutritional immunity. IMPORTANCE Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen causing systemic infections in humans and animals. The competition for iron between ExPEC and the host is a determinant for ExPEC to establish a successful infection. Here, we sought to elucidate the role of transferrin (TF) in the interaction between ExPEC and the host. Our results revealed that holo-TF could be utilized by ExPEC to enhance its growth in culture medium and survival in macrophages. Furthermore, the role of elongation factor G (EFG), a novel holo-TF-binding and TF-iron release protein, was confirmed in this study. Our work provides insights into the iron acquisition mechanism of ExPEC, deepens understanding of the interaction between holo-TF and pathogens, and broadens further researches into the molecular mechanism of ExPEC pathogenicity.