RESUMEN
In most organisms, Cys-tRNA(Cys) is directly synthesized by cysteinyl-tRNA synthetase (CysRS). Many methanogenic archaea, however, use a two-step, indirect pathway to synthesize Cys-tRNA(Cys) owing to a lack of CysRS and cysteine-biosynthesis systems. This reaction is catalyzed by O-phosphoseryl-tRNA synthetase (SepRS), Sep-tRNA:Cys-tRNA synthase (SepCysS) and SepRS/SepCysS pathway enhancer (SepCysE) as the transsulfursome, in which SepCysE connects both SepRS and SepCysS. On the transsulfursome, SepRS first ligates an O-phosphoserine to tRNA(Cys), and the mischarged intermediate Sep-tRNA(Cys) is then transferred to SepCysS, where it is further modified to Cys-tRNA(Cys). In this study, a subcomplex of the transsulfursome with tRNA(Cys) (SepCysS-SepCysE-tRNA(Cys)), which is involved in the second reaction step of the indirect pathway, was constructed and then crystallized. The crystals diffracted X-rays to a resolution of 2.6â Å and belonged to space group P6522, with unit-cell parameters a = b = 107.2, c = 551.1â Å. The structure determined by molecular replacement showed that the complex consists of a SepCysS dimer, a SepCysE dimer and one tRNA(Cys) in the asymmetric unit.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Proteínas Arqueales/química , Methanocaldococcus/química , ARN de Transferencia de Cisteína/química , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Methanocaldococcus/enzimología , Plásmidos/química , Plásmidos/metabolismo , ARN de Transferencia de Cisteína/genética , ARN de Transferencia de Cisteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Difracción de Rayos XRESUMEN
Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The long α-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor.
Asunto(s)
Proteínas Fúngicas/química , ARN Ribosómico 5S/química , Proteínas Ribosómicas/química , Ribosomas/química , Aspergillus nidulans , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , ARN Ribosómico 5S/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
Rpf2 and Rrs1 are essential proteins for ribosome biogenesis. These proteins form a complex (the Rpf2-subcomplex) with 5S rRNA and two ribosomal proteins (L5 and L11). This complex is recruited to the ribosome precursor (the 90S pre-ribosome). This recruitment is necessary for the maturation of 25S rRNA. Genetic depletion of Rpf2 and Rrs1 results in accumulation of the 25S rRNA precursor. In this study, Rpf2 and Rrs1 from Aspergillus nidulans were co-overexpressed in Escherichia coli, purified and crystallized. Subsequent analysis revealed that these crystals contained the central core region of the complex consisting of both N-terminal domains. X-ray diffraction data were collected to 2.35â Å resolution. Preliminary analysis revealed that the crystals belonged to space group P212121, with unit-cell parameters a = 54.1, b = 123.3, c = 133.8â Å. There are two complexes in the asymmetric unit. Structure determination using selenomethionine-labelled protein is in progress.
Asunto(s)
Proteínas Nucleares/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Proteínas de Unión al ARNRESUMEN
Methanogenic archaea lack cysteinyl-tRNA synthetase; they synthesize Cys-tRNA and cysteine in a tRNA-dependent manner. Two enzymes are required: Phosphoseryl-tRNA synthetase (SepRS) forms phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)), which is converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS). This represents the ancestral pathway of Cys biosynthesis and coding in archaea. Here we report a translation factor, SepCysE, essential for methanococcal Cys biosynthesis; its deletion in Methanococcus maripaludis causes Cys auxotrophy. SepCysE acts as a scaffold for SepRS and SepCysS to form a stable high-affinity complex for tRNA(Cys) causing a 14-fold increase in the initial rate of Cys-tRNA(Cys) formation. Based on our crystal structure (2.8-Å resolution) of a SepCysSâ SepCysE complex, a SepRSâ SepCysEâ SepCysS structure model suggests that this ternary complex enables substrate channeling of Sep-tRNA(Cys). A phylogenetic analysis suggests coevolution of SepCysE with SepRS and SepCysS in the last universal common ancestral state. Our findings suggest that the tRNA-dependent Cys biosynthesis proceeds in a multienzyme complex without release of the intermediate and this mechanism may have facilitated the addition of Cys to the genetic code.
Asunto(s)
Archaea/metabolismo , Proteínas Arqueales/metabolismo , Cisteína/biosíntesis , Methanococcus/metabolismo , Factores de Iniciación de Péptidos/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Acetilación , Proteínas Arqueales/química , Secuencia Conservada , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Factores de Iniciación de Péptidos/química , Unión Proteica , Estructura Terciaria de Proteína , ARN de Transferencia de Cisteína/químicaRESUMEN
Removal of anti-association factor, Tif6 (eIF6), by elongation factor-like 1 (EFL1) and Shwachman-Bodian-Diamond syndrome (SBDS) protein is a critical step in the late stage of ribosome maturation. Although EFL1 is known to have GTPase activity that is stimulated by SBDS, how they cooperatively trigger dissociation of Tif6 from the ribosome remains to be elucidated. In the present study, the interaction between EFL1 and SBDS was analyzed by size exclusion chromatography, gel shift assay, and isothermal titration calorimetry (ITC). The results showed that EFL1 interacted directly with SBDS. ITC experiments using domain-truncated mutants showed that the interaction between EFL1 and SBDS is governed by the insertion domain of EFL1 and domains II-III of SBDS. Circular dichroism spectroscopy showed that the insertion domain of EFL1 has a random structure in the absence of SBDS, whereas the disadvantageous entropy change observed on ITC suggested a fixed conformation coupled with complex formation with SBDS. Based on these observations together with those reported previously, we propose roles of EFL1 and SBDS in ribosomal maturation.
Asunto(s)
GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Enfermedades de la Médula Ósea/genética , Enfermedades de la Médula Ósea/metabolismo , Insuficiencia Pancreática Exocrina/genética , Insuficiencia Pancreática Exocrina/metabolismo , GTP Fosfohidrolasas/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Lipomatosis/genética , Lipomatosis/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Factor 2 de Elongación Peptídica/química , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Síndrome de Shwachman-Diamond , TermodinámicaRESUMEN
Additive, indifferent and antagonistic effects were observed in combinations of epigallocatechin gallate (EGCg, a main constituent of tea catechins) with12 non-beta-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The combinations of EGCg with the inhibitors of either protein or nucleic acid synthesis showed additive or indifferent effects. These antibiotics included tetracycline, minocycline, chloramphenicol, streptomycin, gentamicin, kanamycin, erythromycin, rifampicin and ofloxacin. In contrast, EGCg showed an antagonistic tendency against glycopeptide antibiotics (vancomycin, teicoplanin and polymyxin B). The common property of these antibiotics is the peptide backbone structure, suggesting a direct binding of EGCg with the antibiotics. The above results indicate that tea catechins may affect the activities of antibiotics both positively and negatively.
Asunto(s)
Antibacterianos/farmacología , Catequina/análogos & derivados , Catequina/farmacología , Quimioterapia Combinada/farmacología , Resistencia a la Meticilina/fisiología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Catequina/química , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Sinergismo Farmacológico , Quimioterapia Combinada/química , Humanos , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Combinations of carbapenems and epigallocatechin gallate (EGCg; a main constituent of tea catechins) showed potent synergy against 24 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). MICs of imipenem in the presence of EGCg at 3.125, 6.25, 12.5, and 25 microg/ml, were restored to the susceptible breakpoint (< or =4 microg/ml) for 8, 38, 46, and 75% of the MRSA isolates, respectively. Similar results were also observed for combinations of panipenem or meropenem and EGCg. Therefore, the combinations may be worthy of further evaluation in vivo against MRSA infection.
