Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
FEBS Lett ; 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32997337

RESUMO

Aquaporins are transmembrane water channels found in almost every living organism. Numerous studies have brought a good understanding of both water transport through their pores and the regulations taking place at the molecular level, but subtleties remain to be clarified. Recently, a voltage-related gating mechanism involving the conserved arginine of the channel's main constriction was captured for human aquaporins through molecular dynamics studies. With a similar approach, we show that this voltage-gating could be conserved among this family and that the underlying mechanism could explain part of plant AQPs diversity when contextualized to high ionic concentrations provoked by drought. Finally, we identified residues as adaptive traits which constitute good targets for drought resistance plant breeding research.

2.
Plant J ; 2020 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-32639596

RESUMO

Roses use a non-canonical pathway involving a Nudix hydrolase, RhNUDX1, to synthesize their monoterpenes, especially geraniol. Here we report the characterization of another expressed NUDX1 gene from the rose cultivar Rosa x wichurana, RwNUDX1-2. In order to study the function of the RwNUDX1-2 protein, we analyzed the volatile profiles of an F1 progeny generated by crossing R. chinensis cv. 'Old Blush' with R. x wichurana. A correlation test of the volatilomes with gene expression data revealed that RwNUDX1-2 is involved in the biosynthesis of a group of sesquiterpenoids, especially E,E-farnesol, in addition to other sesquiterpenes. In vitro enzyme assays and heterologous in planta functional characterization of the RwNUDX1-2 gene corroborated this result. A quantitative trait locus (QTL) analysis was performed using the data of E,E-farnesol contents in the progeny and a genetic map was constructed based on gene markers. The RwNUDX1-2 gene co-localized with the QTL for E,E-farnesol content, thereby confirming its function in sesquiterpenoid biosynthesis in R. x wichurana. Finally, in order to understand the structural bases for the substrate specificity of rose NUDX proteins, the RhNUDX1 protein was crystallized, and its structure was refined to 1.7 Å. By molecular modeling of different rose NUDX1 protein complexes with their respective substrates, a structural basis for substrate discrimination by rose NUDX1 proteins is proposed.

3.
Biochem J ; 477(2): 445-458, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31808794

RESUMO

The neuroblastoma breakpoint family (NBPF) consists of 24 members that play an important role in neuroblastoma and other cancers. NBPF is an evolutionarily recent gene family that encodes several repeats of Olduvai domain and an abundant N-terminal region. The function and biochemical properties of both Olduvai domain and the N-terminal region remain enigmatic. Human NBPF15 encodes a 670 AA protein consisting of six clades of Olduvai domains. In this study, we synthesized and expressed full-length NBPF15, and purified a range of NBPF15 truncations which were analyzed using dynamic light scattering (DLS), superdex200 (S200), small-angle X-ray scattering (SAXS), far-UV circular dichroism (CD) spectroscopy, transmission electron microscope (TEM), and crystallography. We found that proteins containing both the N-terminal region and Olduvai domain are heterogeneous with multiple types of aggregates, and some of them underwent a liquid-to-solid phase transition, probably because of the entanglement within the N-terminal coiled-coil. Proteins that contain only the Olduvai domain are homogeneous extended monomers, and those with the conserved clade 1 (CON1) have manifested a tendency to crystallize. We suggest that the entanglements between the mosaic disorder-ordered segments in NBPF15 N terminus have triggered the multiple types of aggregates and phase transition of NBPF15 proteins, which could be associated with Olduvai-related cognitive dysfunction diseases.

4.
J Biol Chem ; 294(1): 142-156, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30425099

RESUMO

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveled additional pivotal roles of the LBD protein family beyond defining lateral organ boundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) to 1.9 Å resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimer relied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.


Assuntos
Proteínas de Plantas/química , Fatores de Transcrição/química , Triticum/química , Motivos de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Plantas/genética , Domínios Proteicos , Fatores de Transcrição/genética , Triticum/genética
5.
Biochem Biophys Res Commun ; 504(1): 334-339, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30190128

RESUMO

RNA helicases are almost ubiquitous important enzymes that take part in multiple aspects of RNA metabolism. Prokaryotes encode fewer RNA helicases than eukaryotes, suggesting that individual prokaryotic RNA helicases may take on multiple roles. The specific functions and molecular mechanisms of bacterial DEAH/RHA helicases are poorly understood, and no structures are available of these bacterial enzymes. Here, we report the first crystal structure of the DEAH/RHA helicase HrpB of Escherichia coli in a complex with ADP•AlF4. It showed an atypical globular structure, consisting of two RecA domains, an HA2 domain and an OB domain, similar to eukaryotic DEAH/RHA helicases. Notably, it showed a unique C-terminal extension that has never been reported before. Activity assays indicated that EcHrpB binds RNA but not DNA, and does not exhibit unwinding activity in vitro. Thus, within cells, the EcHrpB may function in helicase activity-independent RNA metabolic processes.


Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , Difosfato de Adenosina/química , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Ácidos Nucleicos/química , Nucleotídeos/química , Estrutura Secundária de Proteína , RNA/química
6.
Structure ; 26(3): 403-415.e4, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29429875

RESUMO

Helicase DHX36 plays essential roles in cell development and differentiation at least partially by resolving G-quadruplex (G4) structures. Here we report crystal structures of the Drosophila homolog of DHX36 (DmDHX36) in complex with RNA and a series of DNAs. By combining structural, small-angle X-ray scattering, molecular dynamics simulation, and single-molecule fluorescence studies, we revealed that positively charged amino acids in RecA2 and OB-like domains constitute an elaborate structural pocket at the nucleic acid entrance, in which negatively charged G4 DNA is tightly bound and partially destabilized. The G4 DNA is then completely unfolded through the 3'-5' translocation activity of the helicase. Furthermore, crystal structures and DNA binding assays show that G-rich DNA is preferentially recognized and in the presence of ATP, specifically bound by DmDHX36, which may cooperatively enhance the G-rich DNA translocation and G4 unfolding. On the basis of these results, a conceptual G4 DNA-resolving mechanism is proposed.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , DNA/química , Drosophila/metabolismo , RNA/química , Animais , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Drosophila/química , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Quadruplex G , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios Proteicos , Desdobramento de Proteína , RNA/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
7.
J Biomol Struct Dyn ; 36(14): 3666-3679, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29057709

RESUMO

The unique eukaryotic-like Ser/Thr protein kinases of Streptococcus pneumoniae, StkP, plays a primary role in the cell division process. It is composed of an intracellular kinase domain, a transmembrane helix and four extracellular PASTA subunits. PASTA domains were shown to interact with cell wall fragments but the key questions related to the molecular mechanism governing ligand recognition remain unclear. To address this issue, the full-length structural model of StkP was generated by combining small-angle X-ray scattering data with the results of computer simulations. Docking and molecular dynamics studies on the generated three-dimensional model structure reveal the possibility of peptidoglycan fragment binding at the hinge regions between PASTA subunits with a preference for a bent hinge between PASTA3 and PASTA4.


Assuntos
Proteínas de Bactérias/química , Modelos Moleculares , Peptidoglicano/química , Proteínas Serina-Treonina Quinases/química , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Relação Estrutura-Atividade
8.
Nucleic Acids Res ; 46(3): 1486-1500, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29202194

RESUMO

The Saccharomyces cerevisiae Pif1 protein (ScPif1p) is the prototypical member of the Pif1 family of DNA helicases. ScPif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and suppresses genome instability at G-quadruplex motifs. Here, we report the crystal structures of a truncated ScPif1p (ScPif1p237-780) in complex with different ssDNAs. Our results have revealed that a yeast-specific insertion domain protruding from the 2B domain folds as a bundle bearing an α-helix, α16. The α16 helix regulates the helicase activities of ScPif1p through interactions with the previously identified loop3. Furthermore, a biologically relevant dimeric structure has been identified, which can be further specifically stabilized by G-quadruplex DNA. Basing on structural analyses and mutational studies with DNA binding and unwinding assays, a potential G-quadruplex DNA binding site in ScPif1p monomers is suggested. Our results also show that ScPif1p uses the Q-motif to preferentially hydrolyze ATP, and a G-rich tract is preferentially recognized by more residues, consistent with previous biochemical observations. These findings provide a structural and mechanistic basis for understanding the multifunctional ScPif1p.


Assuntos
Trifosfato de Adenosina/química , DNA Helicases/química , DNA Fúngico/química , DNA de Cadeia Simples/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Quadruplex G , Expressão Gênica , Hidrólise , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica
9.
FEBS J ; 284(23): 4051-4065, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28986969

RESUMO

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Quadruplex G , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Genéticos , Modelos Moleculares , Conformação de Ácido Nucleico
10.
Nucleic Acids Res ; 45(3): 1539-1552, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180308

RESUMO

The DEAH box helicase Prp43 is a bifunctional enzyme from the DEAH/RHA helicase family required both for the maturation of ribosomes and for lariat intron release during splicing. It interacts with G-patch domain containing proteins which activate the enzymatic activity of Prp43 in vitro by an unknown mechanism. In this work, we show that the activation by G-patch domains is linked to the unique nucleotide binding mode of this helicase family. The base of the ATP molecule is stacked between two residues, R159 of the RecA1 domain (R-motif) and F357 of the RecA2 domain (F-motif). Using Prp43 F357A mutants or pyrimidine nucleotides, we show that the lack of stacking of the nucleotide base to the F-motif decouples the NTPase and helicase activities of Prp43. In contrast the R159A mutant (R-motif) showed reduced ATPase and helicase activities. We show that the Prp43 R-motif mutant induces the same phenotype as the absence of the G-patch protein Gno1, strongly suggesting that the processing defects observed in the absence of Gno1 result from a failure to activate the Prp43 helicase. Overall we propose that the stacking between the R- and F-motifs and the nucleotide base is important for the activity and regulation of this helicase family.


Assuntos
Trifosfato de Adenosina/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Ativação Enzimática , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Nucleotídeos de Pirimidina/química , Nucleotídeos de Pirimidina/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
11.
J Biol Chem ; 292(14): 5909-5920, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28228481

RESUMO

Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a defined three-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Galinhas , Multimerização Proteica , RecQ Helicases/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Cristalografia por Raios X , Domínios Proteicos , Estrutura Quaternária de Proteína , RecQ Helicases/genética , RecQ Helicases/metabolismo
12.
Nat Commun ; 7: 10336, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26831757

RESUMO

Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Moleculares , Fosforilação , Conformação Proteica , Subunidades Proteicas , Transporte Proteico , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Nucleic Acids Res ; 44(6): 2949-61, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26809678

RESUMO

Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity.


Assuntos
Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Bacteroides/química , DNA Helicases/química , DNA de Cadeia Simples/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Polimerase III/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA de Cadeia Simples/metabolismo , Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 11): 1378-83, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26527264

RESUMO

Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified from Escherichia coli BL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space group P6522 in the apo form without pyridoxal 5'-phosphate bound in the active site.


Assuntos
Antiácidos/farmacologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Triptofanase/química , Técnicas de Cultura de Células , Cristalização , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/isolamento & purificação , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Triptofanase/isolamento & purificação , Difração de Raios X
15.
Nucleic Acids Res ; 43(18): 8942-54, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26384418

RESUMO

ScPif1 DNA helicase is the prototypical member of a 5'-to-3' helicase superfamily conserved from bacteria to human and plays various roles in the maintenance of genomic homeostasis. While many studies have been performed with eukaryotic Pif1 helicases, including yeast and human Pif1 proteins, the potential functions and biochemical properties of prokaryotic Pif1 helicases remain largely unknown. Here, we report the expression, purification and biochemical analysis of Pif1 helicase from Bacteroides sp. 3_1_23 (BsPif1). BsPif1 binds to a large panel of DNA substrates and, in particular, efficiently unwinds partial duplex DNAs with 5'-overhang, fork-like substrates, D-loop and flap-like substrates, suggesting that BsPif1 may act at stalled DNA replication forks and enhance Okazaki fragment maturation. Like its eukaryotic homologues, BsPif1 resolves R-loop structures and unwinds DNA-RNA hybrids. Furthermore, BsPif1 efficiently unfolds G-quadruplexes and disrupts nucleoprotein complexes. Altogether, these results highlight that prokaryotic Pif1 helicases may resolve common issues that arise during DNA transactions. Interestingly, we found that BsPif1 is different from yeast Pif1, but resembles more human Pif1 with regard to substrate specificity, helicase activity and mode of action. These findings are discussed in the context of the possible functions of prokaryotic Pif1 helicases in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteroides/enzimologia , DNA Helicases/metabolismo , DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA/química , DNA Helicases/química , DNA Helicases/isolamento & purificação , Quadruplex G , Especificidade por Substrato
16.
Genes Dev ; 29(13): 1432-46, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26159998

RESUMO

In eukaryotes, three of the four ribosomal RNAs (rRNAs)­the 5.8S, 18S, and 25S/28S rRNAs­are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.


Assuntos
Modelos Moleculares , Proteínas Nucleares/química , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , RNA Ribossômico 5S/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Biomed Res Int ; 2015: 931857, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25692149

RESUMO

RNA helicases from the DEAH/RHA family are present in all the processes of RNA metabolism. The function of two helicases from this family, Prp2 and Prp43, is regulated by protein partners containing a G-patch domain. The G-patch is a glycine-rich domain discovered by sequence alignment, involved in protein-protein and protein-nucleic acid interaction. Although it has been shown to stimulate the helicase's enzymatic activities, the precise role of the G-patch domain remains unclear. The role of G-patch proteins in the regulation of Prp43 activity has been studied in the two biological processes in which it is involved: splicing and ribosome biogenesis. Depending on the pathway, the activity of Prp43 is modulated by different G-patch proteins. A particular feature of the structure of DEAH/RHA helicases revealed by the Prp43 structure is the OB-fold domain in C-terminal part. The OB-fold has been shown to be a platform responsible for the interaction with G-patch proteins and RNA. Though there is still no structural data on the G-patch domain, in the current model, the interaction between the helicase, the G-patch protein, and RNA leads to a cooperative binding of RNA and conformational changes of the helicase.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA/metabolismo , Alinhamento de Sequência
18.
PLoS Biol ; 12(5): e1001860, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24823650

RESUMO

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53-MDM2 pathway. This work presents the functional and structural characterization of the Fap7-Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.


Assuntos
Adenilato Quinase/genética , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Nucleosídeo-Trifosfatase/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleosídeo-Trifosfatase/química , Nucleosídeo-Trifosfatase/metabolismo , Pyrococcus abyssi/genética , Pyrococcus abyssi/metabolismo , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
19.
Plant Physiol ; 162(2): 604-15, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23606597

RESUMO

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.


Assuntos
Metiltransferases/genética , Proteínas de Plantas/genética , Pirazinas/metabolismo , Vitis/genética , Vitis/metabolismo , Vinho , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Qualidade dos Alimentos , Regulação da Expressão Gênica de Plantas , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Odorantes , Proteínas de Plantas/metabolismo , Conformação Proteica , Locos de Características Quantitativas , Homologia de Sequência de Aminoácidos
20.
J Biol Chem ; 287(52): 43454-63, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23129767

RESUMO

We report a functional type I toxin-antitoxin (TA) module expressed by a human pathogen, Staphylococcus aureus. TA systems consist of stable toxins and labile antitoxins encoded within small genetic modules widespread in eubacteria and archaea. TA genes provide stress adaptation and protection against DNA loss or invasion. The genes encoding the SprA1 toxic peptide (PepA1) and the SprA1(AS) RNA antitoxin are within a pathogenicity island on opposite strands and possess a 3' overlap. To prevent peptide toxicity during S. aureus growth, PepA1 expression from stable (half-life > 3 h) SprA1 is repressed by elevated amounts of unstable (half-life = ∼10 mn) SprA1(AS). In vivo, PepA1 localizes at the bacterial membrane and triggers S. aureus death. Based on NMR and CD data, its solution structure was solved and is a long bent, interrupted helix. Molecular dynamics simulations indicate that PepA1 compaction and helical content fluctuate in accordance with its cytoplasm or membrane location. When inserted into the S. aureus membrane, the PepA1 conformation switches to a ∼7-nm-long continuous helix, presumably forming pores to alter membrane integrity. PepA1 expression is induced upon acidic and oxidative stresses by reducing SprA1(AS) levels. As an altruistic behavior during infection, some cells may induce the expression of that toxin that would facilitate departure from the host immune cells for spreading.


Assuntos
Membrana Celular , Proteínas de Membrana , Estresse Oxidativo/fisiologia , Peptídeos , Staphylococcus aureus , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Dicroísmo Circular , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA