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1.
Nat Commun ; 11(1): 5003, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024099

RESUMO

Recognition of a start codon by the initiator aminoacyl-tRNA determines the reading frame of messenger RNA (mRNA) translation by the ribosome. In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the initiator Met-tRNAiMet on the ribosome in the later stages of translation initiation, gating entrance into elongation. Leveraging the long residence time of eIF5B on the ribosome recently identified by single-molecule fluorescence measurements, we determine the cryoEM structure of the naturally long-lived ribosome complex with eIF5B and Met-tRNAiMet immediately before transition into elongation. The structure uncovers an unexpected, eukaryotic specific and dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal subunit.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Subunidades Ribossômicas Maiores/metabolismo , Acilação , Anticódon , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Guanosina Difosfato/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Maiores/química , Subunidades Ribossômicas Maiores/genética , Subunidades Ribossômicas Maiores de Eucariotos , Subunidades Ribossômicas Menores de Eucariotos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
2.
Nat Commun ; 11(1): 2794, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493973

RESUMO

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.


Assuntos
RNA de Transferência de Metionina/metabolismo , Riboswitch , S-Adenosilmetionina/metabolismo , Sequência de Bases , Loci Gênicos , Modelos Biológicos , Conformação de Ácido Nucleico , Óperon/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/genética
3.
Nucleic Acids Res ; 47(21): 11368-11386, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31586395

RESUMO

In bacteria, the assembly factors tightly orchestrate the maturation of ribosomes whose competency for protein synthesis is validated by translation machinery at various stages of translation cycle. However, what transpires to the quality control measures when the ribosomes are produced with assembly defects remains enigmatic. In Escherichia coli, we show that 30S ribosomes that harbour assembly defects due to the lack of assembly factors such as RbfA and KsgA display suboptimal initiation codon recognition and bypass the critical codon-anticodon proofreading steps during translation initiation. These premature ribosomes on entering the translation cycle compromise the fidelity of decoding that gives rise to errors during initiation and elongation. We show that the assembly defects compromise the binding of initiation factor 3 (IF3), which in turn appears to license the rapid transition of 30S (pre) initiation complex to 70S initiation complex by tempering the validation of codon-anticodon interaction during translation initiation. This suggests that the premature ribosomes harbouring the assembly defects subvert the IF3 mediated proofreading of cognate initiation codon to enter the translation cycle.


Assuntos
Códon de Iniciação/metabolismo , Fator de Iniciação 3 em Procariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Códon de Iniciação/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas/genética , Multimerização Proteica/fisiologia , RNA de Transferência de Metionina/metabolismo , Ribossomos/genética
4.
Nat Commun ; 10(1): 2640, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201334

RESUMO

One of the responses to stress by eukaryotic cells is the down-regulation of protein synthesis by phosphorylation of translation initiation factor eIF2. Phosphorylation results in low availability of the eIF2 ternary complex (eIF2-GTP-tRNAi) by affecting the interaction of eIF2 with its GTP-GDP exchange factor eIF2B. We have determined the cryo-EM structure of yeast eIF2B in complex with phosphorylated eIF2 at an overall resolution of 4.2 Å. Two eIF2 molecules bind opposite sides of an eIF2B hetero-decamer through eIF2α-D1, which contains the phosphorylated Ser51. eIF2α-D1 is mainly inserted between the N-terminal helix bundle domains of δ and α subunits of eIF2B. Phosphorylation of Ser51 enhances binding to eIF2B through direct interactions of phosphate groups with residues in eIF2Bα and indirectly by inducing contacts of eIF2α helix 58-63 with eIF2Bδ leading to a competition with Met-tRNAi.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , Biossíntese de Proteínas/fisiologia , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fosforilação/fisiologia , Ligação Proteica/fisiologia , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo
5.
Nat Commun ; 10(1): 2136, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086188

RESUMO

Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.


Assuntos
Fator de Iniciação 2B em Eucariotos/ultraestrutura , Fator de Iniciação 2 em Eucariotos/ultraestrutura , RNA de Transferência de Metionina/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Simulação por Computador , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA de Transferência de Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , eIF-2 Quinase/metabolismo
6.
Sci Rep ; 9(1): 5634, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948752

RESUMO

Oxazolidinones are synthetic antibiotics used for treatment of infections caused by Gram-positive bacteria. They target the bacterial protein synthesis machinery by binding to the peptidyl transferase centre (PTC) of the ribosome and interfering with the peptidyl transferase reaction. Cadazolid is the first member of quinoxolidinone antibiotics, which are characterized by combining the pharmacophores of oxazolidinones and fluoroquinolones, and it is evaluated for treatment of Clostridium difficile gastrointestinal infections that frequently occur in hospitalized patients. In vitro protein synthesis inhibition by cadazolid was shown in Escherichia coli and Staphylococcus aureus, including an isolate resistant against linezolid, the prototypical oxazolidinone antibiotic. To better understand the mechanism of inhibition, we determined a 3.0 Å cryo-electron microscopy structure of cadazolid bound to the E. coli ribosome in complex with mRNA and initiator tRNA. Here we show that cadazolid binds with its oxazolidinone moiety in a binding pocket in close vicinity of the PTC as observed previously for linezolid, and that it extends its unique fluoroquinolone moiety towards the A-site of the PTC. In this position, the drug inhibits protein synthesis by interfering with the binding of tRNA to the A-site, suggesting that its chemical features also can enable the inhibition of linezolid-resistant strains.


Assuntos
Oxazolidinonas/metabolismo , Oxazolidinonas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Acetamidas/farmacologia , Antibacterianos/farmacologia , Infecções por Clostridium/tratamento farmacológico , Microscopia Crioeletrônica/métodos , Escherichia coli/metabolismo , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Peptidil Transferases/antagonistas & inibidores , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo
7.
Mitochondrion ; 47: 18-23, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31022467

RESUMO

Mitochondrial DNA variants in the MT-TM (mt-tRNAMet) gene are rare, typically associated with myopathic phenotypes. We identified a novel MT-TM variant resulting in prolonged seizures with childhood-onset myopathy, retinopathy, short stature and elevated CSF lactate associated with bilateral basal ganglia changes on neuroimaging. Muscle biopsy confirmed multiple respiratory chain deficiencies and focal cytochrome c oxidase (COX) histochemical abnormalities. Next-generation sequencing of the mitochondrial genome revealed a novel m.4412G>A variant at high heteroplasmy levels in muscle that fulfils all accepted criteria for pathogenicity including segregation within single muscle fibres, thus broadening the genotypic and phenotypic landscape of mitochondrial tRNA-related disease.


Assuntos
Gânglios da Base , DNA Mitocondrial , Miopatias Mitocondriais , Mutação Puntual , RNA Mitocondrial/genética , RNA de Transferência de Metionina/genética , Convulsões , Gânglios da Base/metabolismo , Gânglios da Base/patologia , Criança , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Humanos , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/metabolismo , Miopatias Mitocondriais/patologia , Miopatias Mitocondriais/fisiopatologia , RNA Mitocondrial/metabolismo , RNA de Transferência de Metionina/metabolismo , Convulsões/genética , Convulsões/metabolismo , Convulsões/patologia , Convulsões/fisiopatologia
8.
Sci Adv ; 5(12): eaay2118, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31903419

RESUMO

Mammalian mitochondrial ribosomes are unique molecular machines that translate 11 leaderless mRNAs; however, it is not clear how mitoribosomes initiate translation, since mitochondrial mRNAs lack untranslated regions. Mitochondrial translation initiation shares similarities with prokaryotes, such as the formation of a ternary complex of fMet-tRNAMet, mRNA and the 28S subunit, but differs in the requirements for initiation factors. Mitochondria have two initiation factors: MTIF2, which closes the decoding center and stabilizes the binding of the fMet-tRNAMet to the leaderless mRNAs, and MTIF3, whose role is not clear. We show that MTIF3 is essential for survival and that heart- and skeletal muscle-specific loss of MTIF3 causes cardiomyopathy. We identify increased but uncoordinated mitochondrial protein synthesis in mice lacking MTIF3, resulting in loss of specific respiratory complexes. Ribosome profiling shows that MTIF3 is required for recognition and regulation of translation initiation of mitochondrial mRNAs and for coordinated assembly of OXPHOS complexes in vivo.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Biossíntese de Proteínas/fisiologia , Animais , Cardiomiopatia Dilatada/genética , Fator de Iniciação 3 em Eucariotos/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
9.
Nucleic Acids Res ; 47(2): 855-867, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30517694

RESUMO

The heterotrimeric eukaryotic translation initiation factor (eIF) 2 plays critical roles in delivering initiator Met-tRNAiMet to the 40S ribosomal subunit and in selecting the translation initiation site. Genetic analyses of patients with MEHMO syndrome, an X-linked intellectual disability syndrome, have identified several unique mutations in the EIF2S3 gene that encodes the γ subunit of eIF2. To gain insights into the molecular consequences of MEHMO syndrome mutations on eIF2 function, we generated a yeast model of the human eIF2γ-I259M mutant, previously identified in a patient with MEHMO syndrome. The corresponding eIF2γ-I318M mutation impaired yeast cell growth and derepressed GCN4 expression, an indicator of defective eIF2-GTP-Met-tRNAiMet complex formation, and, likewise, overexpression of human eIF2γ-I259M derepressed ATF4 messenger RNA translation in human cells. The yeast eIF2γ-I318M mutation also increased initiation from near-cognate start codons. Biochemical analyses revealed a defect in Met-tRNAiMet binding to the mutant yeast eIF2 complexes in vivo and in vitro. Overexpression of tRNAiMet restored Met-tRNAiMet binding to eIF2 in vivo and rescued the growth defect in the eIF2γ-I318M strain. Based on these findings and the structure of eIF2, we propose that the I259M mutation impairs Met-tRNAiMet binding, causing altered control of protein synthesis that underlies MEHMO syndrome.


Assuntos
Epilepsia/genética , Fator de Iniciação 2 em Eucariotos/genética , Genitália/anormalidades , Hipogonadismo/genética , Retardo Mental Ligado ao Cromossomo X/genética , Microcefalia/genética , Mutação , Obesidade/genética , RNA de Transferência de Metionina/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Códon de Iniciação , Fator de Iniciação 2 em Eucariotos/química , Células HEK293 , Humanos , RNA de Transferência de Metionina/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Nucleic Acids Res ; 46(20): 11061-11074, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30239976

RESUMO

In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.


Assuntos
Proteínas Arqueais/fisiologia , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/fisiologia , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , Sequência de Aminoácidos , Archaea/genética , Archaea/metabolismo , Clonagem Molecular , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Iniciação Traducional da Cadeia Peptídica/genética , Fatores de Iniciação de Peptídeos/química , Conformação Proteica , RNA de Transferência de Metionina/metabolismo
11.
Nature ; 560(7717): 263-267, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30089917

RESUMO

Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation1,2. The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4. Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5, necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet-tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome2. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.


Assuntos
Microscopia Crioeletrônica , Mamíferos , Mitocôndrias/ultraestrutura , Iniciação Traducional da Cadeia Peptídica , Animais , Códon de Iniciação/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/ultraestrutura , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , RNA Mitocondrial/química , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA Mitocondrial/ultraestrutura , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , RNA de Transferência de Metionina/ultraestrutura
12.
Cell Mol Life Sci ; 75(23): 4287-4300, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30019215

RESUMO

The initiator tRNA (Met-tRNA i Met ) at the P site of the small ribosomal subunit plays an important role in the recognition of an mRNA start codon. In bacteria, the initiator tRNA carrier, IF2, facilitates the positioning of Met-tRNA i Met on the small ribosomal subunit. Eukarya contain the Met-tRNA i Met carrier, eIF2 (unrelated to IF2), whose carrier activity is inhibited under stress conditions by the phosphorylation of its α-subunit by stress-activated eIF2α kinases. The stress-resistant initiator tRNA carrier, eIF2A, was recently uncovered and shown to load Met-tRNA i Met on the 40S ribosomal subunit associated with a stress-resistant mRNA under stress conditions. Here, we report that eIF2A interacts and functionally cooperates with eIF5B (a homolog of IF2), and we describe the functional domains of eIF2A that are required for its binding of Met-tRNA i Met , eIF5B, and a stress-resistant mRNA. The results indicate that the eukaryotic eIF5B-eIF2A complex functionally mimics the bacterial IF2 containing ribosome-, GTP-, and initiator tRNA-binding domains in a single polypeptide.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , RNA de Transferência de Metionina/metabolismo , eIF-2 Quinase/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fator de Iniciação 2 em Eucariotos/genética , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Humanos , Mutação , Ligação Proteica , Interferência de RNA , RNA de Transferência de Metionina/genética , Homologia de Sequência de Aminoácidos , eIF-2 Quinase/genética
13.
Nucleic Acids Res ; 46(15): 7873-7885, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29931259

RESUMO

Prokaryotic toxin-antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.


Assuntos
Acetiltransferases/genética , Antitoxinas/genética , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/genética , RNA de Transferência de Isoleucina/genética , Acetilação , Acetiltransferases/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas/genética , Processamento de Proteína Pós-Traducional , RNA de Transferência de Isoleucina/metabolismo , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo
14.
Nat Chem Biol ; 14(6): 618-626, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29769736

RESUMO

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.


Assuntos
Biossíntese de Proteínas , RNA de Transferência de Metionina/metabolismo , RNA de Transferência/metabolismo , Algoritmos , Códon , Corantes/química , Eletroporação , Escherichia coli/metabolismo , Corantes Fluorescentes , Cinética , Aprendizado de Máquina , Microscopia de Fluorescência , Microscopia de Vídeo , RNA Mensageiro , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/metabolismo , Imagem Individual de Molécula
15.
FEBS J ; 285(14): 2654-2661, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29775242

RESUMO

Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNAMet -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP.


Assuntos
Metionina tRNA Ligase/química , Metionina/química , Mutação , Proteinose Alveolar Pulmonar/metabolismo , RNA de Transferência de Metionina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Metionina/metabolismo , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/patologia , RNA de Transferência de Metionina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Aminoacilação de RNA de Transferência
16.
Mol Cell ; 69(4): 610-621.e5, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29452640

RESUMO

Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbon units from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Glicina Hidroximetiltransferase/metabolismo , Mitocôndrias/genética , Iniciação Traducional da Cadeia Peptídica , RNA de Transferência de Metionina/química , Serina/química , Animais , Apoptose , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Citosol/metabolismo , Feminino , Glicina Hidroximetiltransferase/antagonistas & inibidores , Glicina Hidroximetiltransferase/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Processamento de Proteína Pós-Traducional , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Serina/genética , Serina/metabolismo , Tetra-Hidrofolatos/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
17.
RNA Biol ; 15(4-5): 604-613, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28914580

RESUMO

Translation begins at AUG, GUG, or UUG codons in bacteria. Start codon recognition occurs in the P site, which may help explain this first-position degeneracy. However, the molecular basis of start codon specificity remains unclear. In this study, we measured the codon dependence of 30S•mRNA•tRNAfMet and 30S•mRNA•tRNAMet complex formation. We found that complex stability varies over a large range with initiator tRNAfMet, following the same trend as reported previously for initiation rate in vivo (AUG > GUG, UUG > CUG, AUC, AUA > ACG). With elongator tRNAMet, the codon dependence of binding differs qualitatively, with virtually no discrimination between GUG and CUG. A unique feature of initiator tRNAfMet is a series of three G-C basepairs in the anticodon stem, which are known to be important for efficient initiation in vivo. A mutation targeting the central of these G-C basepairs causes the mRNA binding specificity pattern to change in a way reminiscent of elongator tRNAMet. Unexpectedly, for certain complexes containing fMet-tRNAfMet, we observed mispositioning of mRNA, such that codon 2 is no longer programmed in the A site. This mRNA mispositioning is exacerbated by the anticodon stem mutation and suppressed by IF2. These findings suggest that both IF2 and the unique anticodon stem of fMet-tRNAfMet help constrain mRNA positioning to set the correct reading frame during initiation.


Assuntos
Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 2 em Procariotos/genética , RNA Mensageiro/genética , RNA de Transferência de Metionina/genética , Fases de Leitura , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Códon de Iniciação , Escherichia coli/metabolismo , Cinética , Mutação , Conformação de Ácido Nucleico , Fator de Iniciação 2 em Procariotos/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência de Metionina/química , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo
18.
Cell Mol Life Sci ; 75(8): 1483-1497, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29103146

RESUMO

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.


Assuntos
Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Células-Tronco Embrionárias Murinas/enzimologia , Placa Neural/enzimologia , RNA de Transferência de Metionina/metabolismo , 5-Metilcitosina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/citologia , Corpos Embrioides/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Metiltransferases/genética , Camundongos , Mitocôndrias/genética , Células-Tronco Embrionárias Murinas/citologia , Placa Neural/citologia , Placa Neural/crescimento & desenvolvimento , Fosforilação Oxidativa , RNA de Transferência de Metionina/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma
19.
J Bacteriol ; 200(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29109187

RESUMO

Type II toxin-antitoxin (TA) systems play a critical role in the establishment and maintenance of bacterial dormancy. They are composed of a protein toxin and its cognate protein antitoxin. They function to regulate growth under conditions of stress, such as starvation or antibiotic treatment. As cellular proteases degrade the antitoxin, which normally binds and neutralizes the toxin, this frees the toxin to act on its cellular targets and arrest bacterial growth. TA systems are of particular concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae (NTHi), as dormancy may lead to chronic infections and failure of antibiotic treatment. Many targets of VapC toxins have not been identified, to date, and this knowledge is crucial to understanding how toxins control the establishment and maintenance of bacterial dormancy. Accordingly, we characterized the target specificity of the VapC toxins from the two paralogous NTHi vapBC TA systems. RNA sequencing and Northern blot analysis revealed that VapC1 and VapC2 cleave tRNAfMet in the anticodon loop. Overexpression of tRNAfMet suppresses VapC toxicity, suggesting that translation inhibition results from the depletion of tRNAfMet These experiments also identified base pairs in the tRNAfMet anticodon stem that play a key role in VapC-specific cleavage of the tRNA. Together these findings suggest the potential for NTHi VapC1 and VapC2 to induce dormancy by sequence-specific cleavage of tRNAfMetIMPORTANCE Bacterial persistence is a significant concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae, as it can result in recurrent and chronic infections. Toxin-antitoxin systems can lead to persistence by causing bacteria to enter a slow-growing state that renders them antibiotic tolerant. Type II toxin components affect a wide variety of bacterial targets in order to elicit dormancy, and for many toxin-antitoxin systems, these mechanisms are not well understood. Thus, in order to understand how vapBC toxin-antitoxin systems cause dormancy, it is crucial to investigate the substrate specificity of VapC toxins. This study identifies the target of the VapC1 and VapC2 toxins from NTHi and takes important steps toward understanding the specificity of these toxins for their tRNA target.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Transferência de Metionina/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endonucleases/genética , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , RNA de Transferência de Metionina/genética , Análise de Sequência de RNA
20.
J Biol Chem ; 293(4): 1425-1438, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29222331

RESUMO

Defective nucleotide modifications of mitochondrial tRNAs have been associated with several human diseases, but their pathophysiology remains poorly understood. In this report, we investigated the pathogenic molecular mechanism underlying a hypertension-associated 4435A→G mutation in mitochondrial tRNAMet The m.4435A→G mutation affected a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon, which is important for the fidelity of codon recognition and stabilization. We hypothesized that the m.4435A→G mutation introduced an m1G37 modification of tRNAMet, altering its structure and function. Primer extension and methylation activity assays indeed confirmed that the m.4435A→G mutation created a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAMet We found that this mutation altered the tRNAMet structure, indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wildtype molecule. We demonstrated that cybrid cell lines carrying the m.4435A→G mutation exhibited significantly decreased efficiency in aminoacylation and steady-state levels of tRNAMet, as compared with those of control cybrids. The aberrant tRNAMet metabolism resulted in variable decreases in mitochondrial DNA (mtDNA)-encoded polypeptides in the mutant cybrids. Furthermore, we found that the m.4435A→G mutation caused respiratory deficiency, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in mutant cybrids. These results demonstrated that an aberrant m1G37 modification of mitochondrial tRNAMet affected the structure and function of its tRNA and consequently altered mitochondrial function. Our findings provide critical insights into the pathophysiology of maternally inherited hypertension, which is manifested by the deficient tRNA nucleotide modification.


Assuntos
DNA Mitocondrial , Hipertensão/genética , Conformação de Ácido Nucleico , Mutação Puntual , RNA de Transferência de Metionina , Linhagem Celular Transformada , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , RNA/genética , RNA/metabolismo , RNA Mitocondrial , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Relação Estrutura-Atividade
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