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1.
Nat Commun ; 12(1): 7102, 2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34876589

RESUMO

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

2.
Mol Cell ; 81(1): 88-103.e6, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33220178

RESUMO

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.


Assuntos
Acetamidas/química , Cicloexilaminas/química , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Regulação Alostérica , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 2B em Eucariotos/genética , Fator de Iniciação 2B em Eucariotos/metabolismo , Células HeLa , Humanos , Fosforilação
3.
Science ; 364(6439): 495-499, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048492

RESUMO

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.


Assuntos
Fator de Iniciação 2B em Eucariotos/antagonistas & inibidores , Fator de Iniciação 2B em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/química , Estresse Fisiológico , Motivos de Aminoácidos , Microscopia Crioeletrônica , Fator de Iniciação 2B em Eucariotos/metabolismo , Humanos , Fosforilação
4.
Mol Cell ; 74(6): 1205-1214.e8, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31080011

RESUMO

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.


Assuntos
Fatores de Iniciação em Eucariotos/química , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
5.
Nature ; 531(7592): 122-5, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26901872

RESUMO

Eukaryotic cells restrict protein synthesis under various stress conditions, by inhibiting the eukaryotic translation initiation factor 2B (eIF2B). eIF2B is the guanine nucleotide exchange factor for eIF2, a heterotrimeric G protein consisting of α-, ß- and γ-subunits. eIF2B exchanges GDP for GTP on the γ-subunit of eIF2 (eIF2γ), and is inhibited by stress-induced phosphorylation of eIF2α. eIF2B is a heterodecameric complex of two copies each of the α-, ß-, γ-, δ- and ε-subunits; its α-, ß- and δ-subunits constitute the regulatory subcomplex, while the γ- and ε-subunits form the catalytic subcomplex. The three-dimensional structure of the entire eIF2B complex has not been determined. Here we present the crystal structure of Schizosaccharomyces pombe eIF2B with an unprecedented subunit arrangement, in which the α2ß2δ2 hexameric regulatory subcomplex binds two γε dimeric catalytic subcomplexes on its opposite sides. A structure-based in vitro analysis by a surface-scanning site-directed photo-cross-linking method identified the eIF2α-binding and eIF2γ-binding interfaces, located far apart on the regulatory and catalytic subcomplexes, respectively. The eIF2γ-binding interface is located close to the conserved 'NF motif', which is important for nucleotide exchange. A structural model was constructed for the complex of eIF2B with phosphorylated eIF2α, which binds to eIF2B more strongly than the unphosphorylated form. These results indicate that the eIF2α phosphorylation generates the 'nonproductive' eIF2-eIF2B complex, which prevents nucleotide exchange on eIF2γ, and thus provide a structural framework for the eIF2B-mediated mechanism of stress-induced translational control.


Assuntos
Fator de Iniciação 2B em Eucariotos/química , Schizosaccharomyces/química , Motivos de Aminoácidos , Sítios de Ligação , Biocatálise , Reagentes para Ligações Cruzadas/química , Cristalografia por Raios X , Fator de Iniciação 2B em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
6.
J Biol Chem ; 287(52): 43950-60, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23091054

RESUMO

Post-transcriptional modifications of the wobble uridine (U34) of tRNAs play a critical role in reading NNA/G codons belonging to split codon boxes. In a subset of Escherichia coli tRNA, this wobble uridine is modified to 5-methylaminomethyluridine (mnm(5)U34) through sequential enzymatic reactions. Uridine 34 is first converted to 5-carboxymethylaminomethyluridine (cmnm(5)U34) by the MnmE-MnmG enzyme complex. The cmnm(5)U34 is further modified to mnm(5)U by the bifunctional MnmC protein. In the first reaction, the FAD-dependent oxidase domain (MnmC1) converts cmnm(5)U into 5-aminomethyluridine (nm(5)U34), and this reaction is immediately followed by the methylation of the free amino group into mnm(5)U34 by the S-adenosylmethionine-dependent domain (MnmC2). Aquifex aeolicus lacks a bifunctional MnmC protein fusion and instead encodes the Rossmann-fold protein DUF752, which is homologous to the methyltransferase MnmC2 domain of Escherichia coli MnmC (26% identity). Here, we determined the crystal structure of the A. aeolicus DUF752 protein at 2.5 Å resolution, which revealed that it catalyzes the S-adenosylmethionine-dependent methylation of nm(5)U in vitro, to form mnm(5)U34 in tRNA. We also showed that naturally occurring tRNA from A. aeolicus contains the 5-mnm group attached to the C5 atom of U34. Taken together, these results support the recent proposal of an alternative MnmC1-independent shortcut pathway for producing mnm(5)U34 in tRNAs.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , tRNA Metiltransferases/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Metilação , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/genética , S-Adenosilmetionina/metabolismo , Homologia de Sequência de Aminoácidos , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
7.
Protein Sci ; 20(7): 1105-13, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21574198

RESUMO

Post-transcriptional modifications of bases within the transfer RNAs (tRNA) anticodon significantly affect the decoding system. In bacteria and eukaryotes, uridines at the wobble position (U34) of some tRNAs are modified to 5-methyluridine derivatives (xm5U). These xm5U34-containing tRNAs read codons ending with A or G, whereas tRNAs with the unmodified U34 are able to read all four synonymous codons of a family box. In Escherichia coli (E.coli), the bifunctional enzyme MnmC catalyzes the two consecutive reactions that convert 5-carboxymethylaminomethyl uridine (cmnm5U) to 5-methylaminomethyl uridine (mnm5U). The C-terminal domain of MnmC (MnmC1) is responsible for the flavin adenine dinucleotide (FAD)-dependent deacetylation of cmnm5U to 5-aminomethyl uridine (nm5U), whereas the N-terminal domain (MnmC2) catalyzes the subsequent S-adenosyl-L-methionine-dependent methylation of nm5U, leading to the final product, mnm5U34. Here, we determined the crystal structure of E.coli MnmC containing FAD, at 3.0 Å resolution. The structure of the MnmC1 domain can be classified in the FAD-dependent glutathione reductase 2 structural family, including the glycine oxidase ThiO, whereas the MnmC2 domain adopts the canonical class I methyltransferase fold. A structural comparison with ThiO revealed the residues that may be involved in cmnm5U recognition, supporting previous mutational analyses. The catalytic sites of the two reactions are both surrounded by conserved basic residues for possible anticodon binding, and are located far away from each other, on opposite sides of the protein. These results suggest that, although the MnmC1 and MnmC2 domains are physically linked, they could catalyze the two consecutive reactions in a rather independent manner.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Complexos Multienzimáticos/química , RNA de Transferência/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
8.
J Mol Biol ; 401(3): 323-33, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20600111

RESUMO

tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.


Assuntos
Adenosina/análogos & derivados , Methanococcaceae/enzimologia , tRNA Metiltransferases/química , Adenosina/química , Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação Proteica , Conformação Proteica , Proteínas de Saccharomyces cerevisiae
9.
Nucleic Acids Symp Ser (Oxf) ; (53): 265-6, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19749362

RESUMO

SELEX is a conventional method to obtain high affinity nucleic acids to target molecules. In this study, high affinity RNA molecules against SRP19 protein were selected by using a randomized library. The primary and predicted secondary structures of the aptamers are different from those of S-domain RNA which is the natural target of SRP19 protein. Comparison of structural features between S-domain RNA and aptamers might enhance our understanding on RNA-protein interaction.


Assuntos
Aptâmeros de Nucleotídeos/química , Partícula de Reconhecimento de Sinal/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/química , Técnica de Seleção de Aptâmeros , Análise de Sequência de RNA
10.
J Struct Biol ; 168(3): 575-81, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19602440

RESUMO

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670-1673]. Here, we report the 1.55 A crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with alpha/beta structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Thermus thermophilus/enzimologia , Vitamina K 2/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
J Mol Biol ; 383(4): 871-84, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18789948

RESUMO

Trm1 catalyzes a two-step reaction, leading to mono- and dimethylation of guanosine at position 26 in most eukaryotic and archaeal tRNAs. We report the crystal structures of Trm1 from Pyrococcus horikoshii liganded with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. The protein comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds, respectively. The methyl moiety of S-adenosyl-l-methionine points toward the invariant Phe27 and Phe140 within a narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the monomethylated tRNA*Trm1 complex prior to the second methylation. Moreover, the catalytic residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.


Assuntos
Estrutura Terciária de Proteína , Pyrococcus horikoshii/enzimologia , tRNA Metiltransferases/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , RNA de Transferência/química , RNA de Transferência/metabolismo , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , tRNA Metiltransferases/genética
12.
J Mol Biol ; 375(4): 1064-75, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18068186

RESUMO

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2'-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 A resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal beta-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the lambda form as its substrate.


Assuntos
Citidina/análogos & derivados , Citidina/química , RNA Arqueal/química , RNA de Transferência/química , tRNA Metiltransferases/química , tRNA Metiltransferases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Metilação , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pyrococcus horikoshii/enzimologia , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , Homologia de Sequência de Aminoácidos , tRNA Metiltransferases/metabolismo
13.
Biochem Biophys Res Commun ; 355(1): 122-8, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17288993

RESUMO

The transcription factor NusA functions in transcriptional regulation involving termination in bacteria. A NusA homolog consisting of only the two KH domains is widely conserved in archaea, but its function remains unknown. We have found that Aeropyrum pernix NusA strongly binds to a certain CU-rich sequence near a termination signal. Our crystal structure of A. pernix NusA revealed that its spatial arrangement is quite similar to that of the KH domains of bacterial NusA. Thus, we consider archaeal NusA to have retained some functions of bacterial NusA, including the ssRNA-binding ability. Remarkable structural differences between archaeal and bacterial NusA exist at the interface with RNAP, in connection with the different NusA-binding sites around the termination signals. Transcriptional termination in archaea could differ from all of the known bacterial and eukaryal mechanisms, in terms of the combination of a bacterial factor and a eukaryal-type RNAP.


Assuntos
Proteínas Arqueais/química , RNA Arqueal/química , RNA Arqueal/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Arqueais/metabolismo , Sequência de Bases , Biotinilação , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/metabolismo
14.
RNA Biol ; 3(3): 115-22, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17114947

RESUMO

The pseudouridine synthase (Psi synthase) TruA catalyzes the conversion of uridine to pseudouridine at positions 38, 39 and/or 40 in the anticodon stem-loop (ASL) of tRNA. We have determined the crystal structure of TruA from Thermus thermophilus HB8 at 2.25 A resolution. TruA and the other (Psi synthases have a completely conserved active site aspartate, which suggests that the members of this enzyme family share a common catalytic mechanism. The T. thermophilus TruA structure reveals the remarkably flexible structural features in the tRNA-binding cleft, which may be responsible for the primary tRNA interaction. In addition, the charged residues occupying the intermediate positions in the cleft may lead the tRNA to the active site for catalysis. Based on the TruB-tRNA complex structure, the T. thermophilus TruA structure reveals that the tRNA probably makes the melting base pairs move into the cleft, and suggests that a conformational change of the substrate tRNA is necessary to facilitate access to the active site aspartate residue, deep within the cleft.


Assuntos
RNA/genética
15.
Biochim Biophys Acta ; 1750(1): 40-7, 2005 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15893507

RESUMO

TTHA1554 was found as a hypothetical protein composed of 95 amino acids in the genome of the extremely thermophilic bacterium, Thermus thermophilus HB8. Proteins homologous to TTHA1554 are conserved in several bacteria and archaea, although their functions are unknown. To investigate the function of TTHA1554, we identified interacting proteins by using a pull-down assay and mass spectrometry. TTHA1329, which is glutamine synthetase, and TTHA1620, a putative aminotransferase, were identified as TTHA1554 binding proteins. The interactions with TTHA1329 and TTHA1620 were validated using in vitro pull-down assays and surface plasmon resonance biosensor assays with recombinant proteins. Since sequence homology analyses suggested that TTHA1620 was a pyridoxal 5'-phosphate-dependent enzyme, such as an aminotransferase, a cystathionine beta-lyase or a cystalysin, putative substrates were investigated. When cystathionine, cystine and S-methylcysteine were used as substrates, pyruvate was produced by TTHA1620. The data revealed that TTHA1620 has cystathionine beta-lyase enzymatic activity. When TTHA1554 was added to the reaction mixtures, the glutamine synthetase and cystathionine beta-lyase enzymatic activities both increased by approximately two-fold. These results indicated that TTHA1554 is a novel protein (we named it GCBP: glutamine synthetase and cystathionine beta-lyase binding protein) that binds to glutamine synthetase and cystathionine beta-lyase.


Assuntos
Proteínas de Bactérias/metabolismo , Glutamato-Amônia Ligase/metabolismo , Liases/metabolismo , Thermus thermophilus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência Conservada , Espectrometria de Massas/métodos , Mapeamento de Interação de Proteínas/métodos , Thermus thermophilus/genética
16.
Protein Sci ; 14(3): 823-7, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15689504

RESUMO

TT1426, from Thermus thermophilus HB8, is a conserved hypothetical protein with a predicted phosphoribosyltransferase (PRTase) domain, as revealed by a Pfam database search. The 2.01 A crystal structure of TT1426 has been determined by the multiwavelength anomalous dispersion (MAD) method. TT1426 comprises a core domain consisting of a central five-stranded beta sheet surrounded by four alpha-helices, and a subdomain in the C terminus. The core domain structure resembles those of the type I PRTase family proteins, although a significant structural difference exists in an inserted 43-residue region. The C-terminal subdomain corresponds to the "hood," which contains a substrate-binding site in the type I PRTases. The hood structure of TT1426 differs from those of the other type I PRTases, suggesting the possibility that TT1426 binds an unknown substrate. The structure-based sequence alignment provides clues about the amino acid residues involved in catalysis and substrate binding.


Assuntos
Proteínas de Bactérias/química , Pentosiltransferases/química , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , Dados de Sequência Molecular , Pentosiltransferases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Thermus thermophilus/genética
17.
J Bacteriol ; 187(1): 135-42, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15601697

RESUMO

The amf gene cluster encodes a probable secretion system for a peptidic morphogen, AmfS, which induces aerial mycelium formation in Streptomyces griseus. Here we examined the transcriptional control mechanism for the promoter preceding amfT (PamfT) directing the transcription of the amfTSBA operon. High-resolution S1 analysis mapped a transcriptional start point at 31 nucleotides upstream of the translational start codon of amfT. Low-resolution analysis showed that PamfT is developmentally regulated in the wild type and completely abolished in an amfR mutant. The -35 region of PamfT contained the consensus sequence for the binding of BldD, a pleiotropic negative regulator for morphological and physiological development in Streptomyces coelicolor A3(2). The cloned bldD locus of S. griseus showed high sequence similarity to the S. coelicolor counterpart. Transcription of bldD occurred constitutively in both the wild type and an A-factor-deficient mutant of S. griseus, which suggests that the regulatory role of BldD is independent of A-factor. The gel retardation assay revealed that purified BldD and AmfR recombinant proteins specifically bind PamfT. Overproduction of BldD in the wild-type cell conferred a bald phenotype (defective in aerial growth and streptomycin production) and caused marked repression of PamfT activity. An amfT-depleted mutant also showed a bald phenotype but PamfT activity was not affected. Both the bldD-overproducing wild-type strain and the amfT mutant were unable to induce aerial growth of an amfS mutant in a cross-feeding assay, which indicates that these strains are defective in the production of an active AmfS peptide. The results overall suggests that two independent regulators, AmfR and BldD, control PamfT activity via direct binding to determine the transcriptional level of the amf operon responsible for the production and secretion of AmfS peptide, which induces the erection of aerial hyphae in S. griseus.


Assuntos
Proteínas de Bactérias/genética , Óperon , Streptomyces griseus/genética , Transcrição Genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Família Multigênica , Fenótipo , Streptomyces griseus/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Biochemistry ; 43(49): 15510-9, 2004 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-15581363

RESUMO

The arylhydrocarbon receptor (AhR) functions as a ligand-activated transcription factor that regulates the transcription of genes encoding xenobiotic metabolizing enzymes and also mediates most of the toxic effects caused by dioxins and polycyclic aromatic hydrocarbons. The cytosolic AhR complex exists as a transcriptionally cryptic complex, consisting of the 90 kDa heat shock protein (HSP90) and the hepatitis B virus X-associated protein 2 (XAP2). The posttranslational modifications, especially phosphorylation, of the cytosolic AhR-HSP90-XAP2 complex are poorly understood, although the phosphorylation of a transcriptionally active heterodimer of AhR and an AhR nuclear translocator is critically involved in AhR function. To reveal the phosphorylation status involved in AhR function, we used mass spectrometry to determine the site-specific phosphorylation of the steady-state cytosolic AhR complex, prepared from Chinese hamster ovary cells stably expressing mouse AhR. We identified phosphorylations of the HSP90 subunits within the AhR complex at Ser225 and Ser254 of HSP90beta and Ser230 of HSP90alpha. By site-directed mutagenesis, these serine residues were substituted with alanine and glutamic acid to elucidate the role of the HSP90beta serine phosphorylations in the AhR function. Immunoprecipitation assays using COS7 transfectants showed that the replacement of Ser225 and Ser254 by Ala, S225/254A, increased the binding affinity for AhR, as compared with the Glu replacement. In a ligand-induced AhR transcription activity assay using Hepa1 transfectants, the S255/254A mutant exhibited more potent transcription activity than the S225/254E mutant, which had activity similar to that of wild-type HSP90beta. These results suggest that the phosphorylations in the charged linker region of the HSP90 molecule modulate the formation of the functional cytosolic AhR complex.


Assuntos
Citosol/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cricetinae , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfoproteínas/metabolismo , Fosforilação , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/isolamento & purificação , Receptores de Hidrocarboneto Arílico/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina/genética , Serina/metabolismo , Transfecção
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