Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Biol Chem ; 290(40): 24391-402, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26272616

RESUMO

The connective polypeptide 1 (CP1) editing domain of leucyl-tRNA synthetase (LeuRS) from various species either harbors a conserved active site to exclude tRNA mis-charging with noncognate amino acids or is evolutionarily truncated or lost because there is no requirement for high translational fidelity. However, human mitochondrial LeuRS (hmtLeuRS) contains a full-length but degenerate CP1 domain that has mutations in some residues important for post-transfer editing. The significance of such an inactive CP1 domain and a translational accuracy mechanism with different noncognate amino acids are not completely understood. Here, we identified the essential role of the evolutionarily divergent CP1 domain in facilitating hmtLeuRS's catalytic efficiency and endowing enzyme with resistance to AN2690, a broad-spectrum drug acting on LeuRSs. In addition, the canonical core of hmtLeuRS is not stringent for noncognate norvaline (Nva) and valine (Val). hmtLeuRS has a very weak tRNA-independent pre-transfer editing activity for Nva, which is insufficient to remove mis-activated Nva. Moreover, hmtLeuRS chimeras fused with a functional CP1 domain from LeuRSs of other species, regardless of origin, showed restored post-transfer editing activity and acquired fidelity during aminoacylation. This work offers a novel perspective on the role of the CP1 domain in optimizing aminoacylation efficiency.


Assuntos
Aminoacil-tRNA Sintetases/química , Leucina-tRNA Ligase/química , Sequência de Aminoácidos , Aminoácidos/química , Aminoacil-tRNA Sintetases/fisiologia , Aminoacilação , Domínio Catalítico , Dicroísmo Circular , Humanos , Hidrólise , Leucina-tRNA Ligase/fisiologia , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Edição de RNA , Homologia de Sequência de Aminoácidos , Valina/análogos & derivados , Valina/química
2.
Biochemistry ; 46(21): 6258-67, 2007 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-17474713

RESUMO

Protein synthesis and its fidelity rely upon the aminoacyl-tRNA synthetases. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) have evolved a discrete editing domain called CP1 that hydrolyzes the respective incorrectly misaminoacylated noncognate amino acids. Although active CP1 domain fragments have been isolated for IleRS and ValRS, previous reports suggested that the LeuRS CP1 domain required idiosyncratic adaptations to confer editing activity independent of the full-length enzyme. Herein, characterization of a series of rationally designed Escherichia coli LeuRS fragments showed that the beta-strands, which link the CP1 domain to the aminoacylation core of LeuRS, are required for editing of mischarged tRNALeu. Hydrolytic activity was also enhanced by inclusion of short flexible peptides that have been called "hinges" at the end of both LeuRS beta-strands. We propose that these long beta-strand extensions of the LeuRS CP1 domain interact specifically with the tRNA for post-transfer editing of misaminoacylated amino acids.


Assuntos
Leucina-tRNA Ligase/química , Edição de RNA , Motivos de Aminoácidos , Proteínas de Escherichia coli , Hidrólise , Leucina-tRNA Ligase/fisiologia , Fragmentos de Peptídeos , Engenharia de Proteínas
3.
J Mol Biol ; 367(2): 384-94, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17270210

RESUMO

Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.


Assuntos
Leucina-tRNA Ligase/química , Proteínas Mitocondriais/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Teste de Complementação Genética , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Mutação , Peptídeos/química , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Aminoacilação de RNA de Transferência
4.
RNA ; 13(1): 15-21, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17095543

RESUMO

Leucyl-, isoleucyl-, and valyl-tRNA synthetases form a subgroup of related aminoacyl-tRNA synthetases that attach similar amino acids to their cognate tRNAs. To prevent amino acid misincorporation during translation, these enzymes also hydrolyze mischarged tRNAs through a post-transfer editing mechanism. Here we show that LeuRS from the deep-branching bacterium Aquifex aeolicus edits the complete set of aminoacylated tRNAs generated by the three enzymes: Ile-tRNA(Ile), Val-tRNA(Ile), Val-tRNA(Val), Thr-tRNA(Val), and Ile-tRNA(Leu). This unusual enlarged editing property was studied in a model of a primitive editing system containing a composite minihelix carrying the triple leucine, isoleucine, and valine identity mimicking the primitive tRNA precursor. We found that the freestanding LeuRS editing domain can edit this precursor in contrast to IleRS and ValRS editing domains. These results suggest that A. aeolicus LeuRS carries editing properties that seem more primitive than those of IleRS and ValRS. They suggest that the A. aeolicus editing domain has preserved the ambiguous editing property from the ancestral common editing domain or, alternatively, that this plasticity results from a specific metabolic adaptation.


Assuntos
Bactérias/enzimologia , Leucina-tRNA Ligase/fisiologia , Edição de RNA , RNA Bacteriano/metabolismo , Aminoacilação de RNA de Transferência , Bactérias/genética , Sequência de Bases , Evolução Molecular , Isoleucina-tRNA Ligase/química , Isoleucina-tRNA Ligase/fisiologia , Leucina-tRNA Ligase/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Especificidade por Substrato , Valina-tRNA Ligase/química , Valina-tRNA Ligase/fisiologia
5.
J Biol Chem ; 282(6): 3680-7, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17158871

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNA(Pro) aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable K(D) of about 0.3-0.9 microm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNA(Lys) over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS.


Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Proteínas Arqueais/fisiologia , Methanobacteriaceae/enzimologia , Aminoacil-tRNA Sintetases/isolamento & purificação , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Catálise , Leucina-tRNA Ligase/isolamento & purificação , Leucina-tRNA Ligase/metabolismo , Leucina-tRNA Ligase/fisiologia , Lisina-tRNA Ligase/isolamento & purificação , Lisina-tRNA Ligase/metabolismo , Lisina-tRNA Ligase/fisiologia , Methanobacteriaceae/fisiologia , Mapeamento de Interação de Proteínas , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-Híbrido
6.
J Biol Chem ; 280(28): 26099-104, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15917221

RESUMO

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNA(Pro) charging showed that the catalytic efficiency (k(cat)/K(m)) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the K(m) for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS.


Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Leucina-tRNA Ligase/fisiologia , RNA de Transferência de Prolina/química , Aminoacil-tRNA Sintetases/química , Cromatografia , Cromatografia em Gel , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Biblioteca Gênica , Histidina/química , Cinética , Leucina-tRNA Ligase/química , Methanobacteriaceae/metabolismo , Fenótipo , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Temperatura , Técnicas do Sistema de Duplo-Híbrido
7.
Proc Natl Acad Sci U S A ; 97(25): 13743-8, 2000 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-11087829

RESUMO

Mitochondrial leucyl-tRNA synthetase (LeuRS) in the yeast Saccharomyces cerevisiae provides two essential functions. In addition to aminoacylation, LeuRS functions in RNA splicing. The details of how it came to act in splicing are not known. Here we show that Mycobacterium tuberculosis and human mitochondrial LeuRSs can substitute in splicing for the S. cerevisiae mitochondrial LeuRS. Mutations of yeast mitochondrial LeuRS that had previously been shown to abolish splicing activity also eliminate splicing by the M. tuberculosis enzyme. These results suggest the role of LeuRS in splicing in yeast mitochondria results from features of the enzyme that are broadly conserved in evolution. These features are not likely to be designed for splicing per se, but instead have been adopted in yeast for that purpose.


Assuntos
Leucina-tRNA Ligase/fisiologia , Splicing de RNA/fisiologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Clonagem Molecular , Teste de Complementação Genética , Humanos , Íntrons , Leucina-tRNA Ligase/química , Leucina-tRNA Ligase/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos
8.
Nucleic Acids Res ; 14(19): 7529-39, 1986 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-3534789

RESUMO

Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.


Assuntos
Aminoacil-tRNA Sintetases/fisiologia , Isoleucina/metabolismo , Leucina-tRNA Ligase/fisiologia , Leucina/metabolismo , Biossíntese de Proteínas , Monofosfato de Adenosina/biossíntese , Escherichia coli/enzimologia , Homocisteína/metabolismo , Hidrólise , Lactonas/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Saccharomyces cerevisiae/enzimologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...