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1.
Proc Natl Acad Sci U S A ; 111(20): 7284-9, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24799711

RESUMO

The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form essential components of the complexes involved in oxidative phosphorylation or ATP generation for the eukaryotic cell. The mammalian 55S mitoribosome contains significantly smaller rRNAs and a large mass of mitochondrial ribosomal proteins (MRPs), including large mito-specific amino acid extensions and insertions in MRPs that are homologous to bacterial ribosomal proteins and an additional 35 mito-specific MRPs. Here we present the cryo-EM structure analysis of the small (28S) subunit (SSU) of the 55S mitoribosome. We find that the mito-specific extensions in homologous MRPs generally are involved in inter-MRP contacts and in contacts with mito-specific MRPs, suggesting a stepwise evolution of the current architecture of the mitoribosome. Although most of the mito-specific MRPs and extensions of homologous MRPs are situated on the peripheral regions, they also contribute significantly to the formation of linings of the mRNA and tRNA paths, suggesting a tailor-made structural organization of the mito-SSU for the recruitment of mito-specific mRNAs, most of which do not possess a 5' leader sequence. In addition, docking of previously published coordinates of the large (39S) subunit (LSU) into the cryo-EM map of the 55S mitoribosome reveals that mito-specific MRPs of both the SSU and LSU are involved directly in the formation of six of the 15 intersubunit bridges.


Assuntos
Mitocôndrias/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Animais , Sítios de Ligação , Bovinos , Microscopia Crioeletrônica , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Processamento de Imagem Assistida por Computador , Fígado/metabolismo , Conformação Proteica , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/metabolismo
2.
Proc Natl Acad Sci U S A ; 108(10): 3918-23, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21368145

RESUMO

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.


Assuntos
Eubacterium/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Aminoácidos/química , Animais , Bovinos , Microscopia Crioeletrônica , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Modelos Moleculares , Fosforilação Oxidativa , Ribossomos/metabolismo
3.
Biochim Biophys Acta ; 1819(9-10): 1035-54, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22172991

RESUMO

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.


Assuntos
Mitocôndrias , Proteínas Mitocondriais/biossíntese , Fator de Iniciação 2 em Procariotos , RNA Mensageiro , Animais , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fator de Iniciação 2 em Procariotos/genética , Fator de Iniciação 2 em Procariotos/metabolismo , Biossíntese de Proteínas , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo
4.
Biochim Biophys Acta ; 1814(12): 1779-84, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22015679

RESUMO

Mammalian mitochondrial translational initiation factor 3 (IF3(mt)) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3(mt) binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3(mt) cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3(mt) has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3(mt) except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.


Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Animais , Bovinos , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/genética , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Biológicos , Modelos Moleculares , Iniciação Traducional da Cadeia Peptídica/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/genética
5.
J Biol Chem ; 285(36): 28379-86, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20610392

RESUMO

Mammalian mitochondrial mRNAs are basically leaderless, having few or no untranslated nucleotides prior to the 5'-start codon. We demonstrate here that mammalian mitochondrial 55 S ribosomes preferentially form initiation complexes at a 5'-terminal AUG codon over an internal AUG. The preferential use of the 5'-start codon is also seen on mitochondrial 28 S small subunits, which suggests that mitochondrial translation initiation on leaderless mRNAs does not require the large ribosomal subunit. The selection of the 5'-AUG is dependent on the presence of fMet-tRNA and is enhanced by the presence of the mitochondrial initiation factor IF2(mt). In prokaryotes, IF3 is believed to antagonize initiation on leaderless mRNAs. However, IF3(mt) stimulates initiation complex formation on leaderless mRNAs when tested with 55 S ribosomes. The addition of even a few nucleotides 5' to the AUG codon significantly reduces the efficiency of initiation, highlighting the importance of the leaderless or nearly leaderless nature of mitochondrial mRNAs. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of tRNA(Met) attached at the 5'-end of a mitochondrial protein-coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.


Assuntos
Códon de Iniciação/metabolismo , Mitocôndrias/metabolismo , Ribossomos/metabolismo , Animais , Sequência de Bases , Bovinos , Códon de Iniciação/genética , Humanos , Mitocôndrias/genética , Dados de Sequência Molecular , Fosfatos/metabolismo , Biossíntese de Proteínas , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA Ribossômico 28S/genética , RNA Ribossômico 28S/metabolismo , Ribonucleotídeos/metabolismo
6.
J Biol Chem ; 285(45): 34991-8, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20739282

RESUMO

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica/fisiologia , Ribossomos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/genética , Humanos , Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Mapeamento de Peptídeos/métodos , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribossomos/química
7.
J Biol Chem ; 285(36): 28353-62, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20601428

RESUMO

In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal approximately 100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K(d) of 0.3-0.8 microM and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades Ribossômicas Maiores/metabolismo
8.
Biochim Biophys Acta ; 1802(7-8): 692-8, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20435138

RESUMO

Mammalian mitochondria synthesize a set of thirteen proteins that are essential for energy generation via oxidative phosphorylation. The genes for all of the factors required for synthesis of the mitochondrially encoded proteins are located in the nuclear genome. A number of disease-causing mutations have been identified in these genes. In this manuscript, we have elucidated the mechanisms of translational failure for two disease states characterized by lethal mutations in mitochondrial elongation factor Ts (EF-Ts(mt)) and elongation factor Tu (EF-Tu(mt)). EF-Tu(mt) delivers the aminoacyl-tRNA (aa-tRNA) to the ribosome during the elongation phase of protein synthesis. EF-Ts(mt) regenerates EF-Tu(mt):GTP from EF-Tu(mt):GDP. A mutation of EF-Ts(mt) (R325W) leads to a two-fold reduction in its ability to stimulate the activity of EF-Tu(mt) in poly(U)-directed polypeptide chain elongation. This loss of activity is caused by a significant reduction in the ability of EF-Ts(mt) R325W to bind EF-Tu(mt), leading to a defect in nucleotide exchange. A mutation of Arg336 to Gln in EF-Tu(mt) causes infantile encephalopathy caused by defects in mitochondrial translation. EF-Tu(mt) R336Q is as active as the wild-type protein in polymerization using Escherichia coli 70S ribosomes and E. coli [(14)C]Phe-tRNA but is inactive in polymerization with mitochondrial [(14)C]Phe-tRNA and mitochondrial 55S ribosomes. The R336Q mutation causes a two-fold decrease in ternary complex formation with E. coli aa-tRNA but completely inactivates EF-Tu(mt) for binding to mitochondrial aa-tRNA. Clearly the R336Q mutation in EF-Tu(mt) has a far more drastic effect on its interaction with mitochondrial aa-tRNAs than bacterial aa-tRNAs.


Assuntos
Genes Letais , Mitocôndrias/metabolismo , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/fisiologia , Biossíntese de Proteínas/genética , Substituição de Aminoácidos/fisiologia , Animais , Bovinos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Genes Letais/fisiologia , Mitocôndrias/genética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Mutação/fisiologia , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/fisiologia , Fatores de Alongamento de Peptídeos/análise , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Multimerização Proteica , RNA de Transferência Aminoácido-Específico/metabolismo , Relação Estrutura-Atividade
9.
Nucleic Acids Res ; 37(5): 1616-27, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19151083

RESUMO

Mitochondrial (mt) tRNA(Met) has the unusual modified nucleotide 5-formylcytidine (f(5)C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNA(Met) carrying f(5)C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNA(Met) lacking f(5)C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNA(Met)(m) with the anticodon ac(4)CAU (ac(4)C = 4-acetylcytidine) and the bovine cytoplasmic initiator tRNA(Met) (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f(5)C modification in mt tRNA(Met) plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f(5)C-G and f(5)C-A based on the chemical properties of f(5)C are presented.


Assuntos
Códon/química , Citidina/análogos & derivados , Mitocôndrias/genética , Biossíntese de Proteínas , RNA de Transferência de Metionina/química , RNA/química , Animais , Anticódon/química , Pareamento de Bases , Sequência de Bases , Bovinos , Códon de Iniciação/química , Citidina/química , Escherichia coli/genética , Metionina/metabolismo , Dados de Sequência Molecular , RNA/metabolismo , RNA Mitocondrial , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
10.
Biochem Biophys Res Commun ; 391(1): 942-6, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19962967

RESUMO

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2(mt)) and mitochondrial initiation factor 3 (IF3(mt)). This observation indicates that IF2(mt) and IF3(mt), while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3(mt) dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2(mt). The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.


Assuntos
Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/biossíntese , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Espermina/farmacologia , Animais , Bovinos , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Mitocôndrias/metabolismo , RNA de Transferência de Metionina/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
11.
RNA ; 14(5): 862-71, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18367717

RESUMO

The mammalian mitochondrial genome encodes 13 proteins, which are synthesized at the direction of nine monocistronic and two dicistronic mRNAs. These mRNAs lack both 5' and 3' untranslated regions. The mechanism by which the specialized mitochondrial translational apparatus locates start codons and initiates translation of these leaderless mRNAs is currently unknown. To better understand this mechanism, the secondary structures near the start codons of all 13 open reading frames have been analyzed using RNA SHAPE chemistry. The extent of structure in these mRNAs as assessed experimentally is distinctly lower than would be predicted by current algorithms based on free energy minimization alone. We find that the 5' ends of all mitochondrial mRNAs are highly unstructured. The first 35 nucleotides for all mitochondrial mRNAs form structures with free energies less favorable than -3 kcal/mol, equal to or less than a single typical base pair. The start codons, which lie at the very 5' ends of these mRNAs, are accessible within single stranded motifs in all cases, making them potentially poised for ribosome binding. These data are consistent with a model in which the specialized mitochondrial ribosome preferentially allows passage of unstructured 5' sequences into the mRNA entrance site to participate in translation initiation.


Assuntos
Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , Animais , Sequência de Bases , Bovinos , Códon de Iniciação/genética , Primers do DNA/genética , Complexo I de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Perfilação da Expressão Gênica , Genes , Genoma Mitocondrial , Modelos Genéticos , Modelos Moleculares , NADH Desidrogenase/genética , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/metabolismo , RNA Mitocondrial
12.
Nucleic Acids Res ; 36(2): 589-97, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18056078

RESUMO

Mammalian mitochondrial initiation factor 3 (IF3(mt)) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3(mt) to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K(d) for the binding of IF3(mt) to 28S subunits is approximately 30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3(mt) has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K(d) = 1.5 muM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K(d) = 0.12-0.25 muM). IF3(mt) does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3(mt) is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3(mt) from the 28S subunits upon 39S subunit joining.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Animais , Bovinos , Fatores de Iniciação em Eucariotos/genética , Evolução Molecular , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Fator de Iniciação 3 em Procariotos/química , Ligação Proteica , RNA de Transferência de Metionina/metabolismo , Deleção de Sequência , Ressonância de Plasmônio de Superfície
13.
Nat Commun ; 11(1): 3830, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737313

RESUMO

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-G1mt) 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-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. 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.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Elongação Traducional da Cadeia Peptídica , Fator G para Elongação de Peptídeos/química , RNA Mitocondrial/química , RNA de Transferência/química , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Microscopia Crioeletrônica , Células HEK293 , Humanos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
14.
Biochemistry ; 48(15): 3269-78, 2009 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-19239245

RESUMO

Mitochondrial translational initiation factor 3 (IF3(mt)) is a 29 kDa protein that has N- and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3(mt) important for its activity. On the basis of homology models of IF3(mt), mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170-171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with K(d) values similar to that of the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3(mt) to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3(mt) to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3(mt) plays an active role in initiation of translation.


Assuntos
Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Biossíntese de Proteínas , Animais , Bovinos , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fatores de Iniciação em Eucariotos/fisiologia , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética , Humanos , Camundongos , Proteínas Mitocondriais/fisiologia , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fator de Iniciação 3 em Procariotos/química , Fator de Iniciação 3 em Procariotos/genética , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína
16.
iScience ; 12: 76-86, 2019 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-30677741

RESUMO

The human mitochondrial translational initiation factor 3 (IF3mt) carries mitochondrial-specific amino acid extensions at both its N and C termini (N- and C-terminal extensions [NTE and CTE, respectively]), when compared with its eubacterial counterpart. Here we present 3.3- to 3.5-Å-resolution cryoelectron microscopic structures of the mammalian 28S mitoribosomal subunit in complex with human IF3mt. Unique contacts observed between the 28S subunit and N-terminal domain of IF3mt explain its unusually high affinity for the 28S subunit, whereas the position of the mito-specific NTE suggests NTE's role in binding of initiator tRNA to the 28S subunit. The location of the C-terminal domain (CTD) clarifies its anti-association activity, whereas the orientation of the mito-specific CTE provides a mechanistic explanation for its role in destabilizing initiator tRNA in the absence of mRNA. Furthermore, our structure hints at a possible role of the CTD in recruiting leaderless mRNAs for translation initiation. Our findings highlight unique features of IF3mt in mitochondrial translation initiation.

17.
Mitochondrion ; 8(3): 254-61, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18539099

RESUMO

Mutations in mitochondrial small subunit ribosomal proteins MRPS16 or MRPS22 cause severe, fatal respiratory chain dysfunction due to impaired translation of mitochondrial mRNAs. The loss of either MRPS16 or MRPS22 was accompanied by the loss of most of another small subunit protein MRPS11. However, MRPS2 was reduced only about 2-fold in patient fibroblasts. This observation suggests that the small ribosomal subunit is only partially able to assemble in these patients. Two large subunit ribosomal proteins, MRPL13 and MRPL15, were present in substantial amounts suggesting that the large ribosomal subunit is still present despite a non-functional small subunit.


Assuntos
Mitocôndrias/genética , Proteínas Mitocondriais/genética , RNA Ribossômico 16S/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/fisiologia , Proteínas Mitocondriais/fisiologia , Modelos Moleculares , Mutação , Proteínas Ribossômicas/fisiologia , Subunidades Ribossômicas Maiores/metabolismo , Subunidades Ribossômicas Menores/metabolismo
18.
Mitochondrion ; 39: 1-8, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28804013

RESUMO

Initiation factor 3 (IF3) is a conserved translation factor. Mutations in mitochondrial IF3 (IF3mt) have been implicated in disease pathology. Escherichia coli infCΔ55, compromised for IF3 activity, has provided an excellent heterologous system for IF3mt structure-function analysis. IF3mt allowed promiscuous initiation from AUA, AUU and ACG codons but avoided initiation with initiator tRNAs lacking the conserved 3GC pairs in their anticodon stems. Expression of IF3mt N-terminal domain, or IF3mt devoid of its typical N-, and C-terminal extensions improved fidelity of initiation in E. coli. The observations suggest that the IF3mt terminal extensions relax the fidelity of translational initiation in mitochondria.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas Mitocondriais/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fator de Iniciação 3 em Procariotos/metabolismo , Códon de Iniciação , Humanos , Proteínas Mitocondriais/genética , Fator de Iniciação 3 em Procariotos/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Methods Enzymol ; 430: 59-78, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17913635

RESUMO

Two mammalian mitochondrial initiation factors have been identified. Initiation factor 2 (IF2(mt)) selects the initiator tRNA (fMet-tRNA) and promotes its binding to the ribosome. Initiation factor 3 (IF3(mt)) promotes the dissociation of the 55S mitochondrial ribosome into subunits and may play additional, less-well-understood, roles in initiation complex formation. Native bovine IF2(mt) was purified from liver a number of years ago. The yield of this factor is very low making biochemical studies difficult. The cDNA for bovine IF2(mt) was expressed in Escherichia coli under the control of the T7 polymerase promoter in a vector that provides a His(6)-tag at the C-terminus of the expressed protein. This factor was expressed in E. coli and purified by chromatography on Ni-NTA resins. The expressed protein has a number of degradation products in partially purified preparations and this factor is then further purified by high-performance liquid chromatography or gravity chromatography on anion exchange resins. IF3(mt) has never been purified from any mammalian system. However, the cDNA for this protein can be identified in the expressed sequence tag (EST) libraries. The portion of the sequence encoding the region of human IF3(mt) predicted to be present in the mitochondrially imported form of this factor was cloned and expressed in E. coli using a vector that provides a C-terminal His(6)-tag. The tagged factor is partially purified on Ni-NTA resins. However, a major proteolytic fragment arising from a defined cleavage of this protein is present in these preparations. This contaminant can be removed by a single step of high-performance liquid chromatography on a cation exchange resin. Alternatively, the mature form of IF3(mt) can be purified by two sequential passes through a gravity S-Sepharose column.


Assuntos
Fator de Iniciação 2 em Eucariotos , Fator de Iniciação 3 em Eucariotos , Animais , Bovinos , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/isolamento & purificação , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/isolamento & purificação , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , RNA de Transferência de Metionina/isolamento & purificação , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
20.
Mol Biochem Parasitol ; 152(2): 203-12, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17292489

RESUMO

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.


Assuntos
Leishmania/química , Proteínas Mitocondriais/química , Proteínas de Protozoários/química , Ribonucleoproteínas/química , Animais , Microscopia Crioeletrônica , Leishmania/metabolismo , Leishmania/ultraestrutura , Mitocôndrias/metabolismo , Proteínas Mitocondriais/isolamento & purificação , Proteínas Mitocondriais/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteômica , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/ultraestrutura , RNA Ribossômico/química , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/ultraestrutura , Proteínas Ribossômicas/química , Espectrometria de Massas em Tandem
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