RESUMEN
Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.
Asunto(s)
Saccharomyces cerevisiae , Codón de Terminación/genética , Codón de Terminación/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Aeromonas salmonicida/genética , Ingeniería de Proteínas , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/genética , ARN de Transferencia de Cisteína/metabolismo , Humanos , Conformación de Ácido NucleicoRESUMEN
Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNACys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNACys can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNACys in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions -1 to -4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.
Asunto(s)
Aminoácidos , ARN de Transferencia de Cisteína , Aminoácidos/metabolismo , Escherichia coli/genética , Biosíntesis de Proteínas , Proteínas/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia de Cisteína/genética , ARN de Transferencia de Cisteína/metabolismo , Ribosomas/metabolismoRESUMEN
OBJECTIVES: Recent studies have shown that tRNA-derived RNA fragments (tRFs) are novel regulators of post-transcriptional gene expression. However, the expression profiles and their role in post-transcriptional gene regulation in chondrocytes is unknown. Here, we determined tRFs expression profile and explored tRF-3003a role in post-transcriptional gene regulation in IL-1ß stimulated chondrocytes. METHODS: We used qPCR arrays to determine tRNAs and tRFs expression in age- and sex-matched primary human OA chondrocytes and TC28/I2 cells stimulated with IL-1ß. Chondrocytes were transfected with tRNA-CysGCA overexpression plasmid or tRF-3003a mimic and 3'UTR luciferase reporter plasmids of mRNAs harboring predicted tRF target "seed sequence". The AGO-RNA-induced silencing complex (AGO-RISC)-dependent repressive activity of tRF-3003a was determined by siRNA-mediated knockdown of AGO2. RESULTS: IL-1ß increased the expression levels of specific tRNAs and of tRF-3003a, a type 3 tRF produced by the cleavage of tRNA-CysGCA. tRF-3003a "seed sequence" was identified in the 3'UTR of JAK3 mRNA and tRNA-CysGCA overexpression or transfection of a tRF-3003a mimic in chondrocytes downregulated JAK3 expression and significantly reduced the activity of the 3'UTR reporter. RIP assay showed enrichment of tRF-3003a into AGO2/RISC in IL-1ß treated chondrocytes. The suppressive effect of tRF-3003a on JAK3 3'UTR reporter was abrogated with siRNA-mediated depletion of AGO2. CONCLUSIONS: We demonstrate that under pathological conditions chondrocytes display perturbations in the expression profile of specific tRNAs and tRFs. Furthermore, a specific tRF namely tRF-3003a can post-transcriptionally regulate JAK3 expression via AGO/RISC formation in chondrocytes. Identification of this novel mechanism may be of value in the design of precision therapies for OA.
Asunto(s)
Condrocitos/metabolismo , Regulación de la Expresión Génica , Osteoartritis/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/genética , ARN de Transferencia de Cisteína/genética , Regiones no Traducidas 3' , Proteínas Argonautas , Línea Celular , Condrocitos/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Janus Quinasa 3/genética , Osteoartritis/metabolismo , Cultivo Primario de Células , ARN Mensajero/efectos de los fármacos , ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Cisteína/metabolismoRESUMEN
tRNA synthetases are responsible for decoding the molecular information, from codons to amino acids. Seryl-tRNA synthetase (SerRS), besides the five isoacceptors of tRNASer, recognizes tRNA[Ser]Sec for the incorporation of selenocysteine (Sec, U) into selenoproteins. The selenocysteine synthesis pathway is known and is dependent on several protein-protein and protein-RNA interactions. Those interactions are not fully described, in particular, involving tRNA[Ser]Sec and SerRS. Here we describe the molecular interactions between the Escherichia coli Seryl-tRNA synthetase (EcSerRS) and tRNA[Ser]Sec in order to determine their specificity, selectivity and binding order, leading to tRNA aminoacylation. The dissociation constant of EcSerRS and tRNA[Ser]Sec was determined as (126 ± 20) nM. We also demonstrate that EcSerRS binds initially to tRNA[Ser]Sec in the presence of ATP for further recognition by E. coli selenocysteine synthetase (EcSelA) for Ser to Sec conversion. The proposed studies clarify the mechanism of tRNA[Ser]Sec incorporation in Bacteria as well as of other domains of life.
Asunto(s)
Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN de Transferencia Aminoácido-Específico/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Serina-ARNt Ligasa/metabolismo , Transferasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Escherichia coli/genética , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Cisteína/genética , Serina-ARNt Ligasa/genética , Termodinámica , Aminoacilación de ARN de Transferencia/genética , Transferasas/genéticaRESUMEN
A Chinese family with matrilineally inherited obesity was assessed and its clinical, genetic, and molecular profiling was conducted. Obesity was observed in matrilineal relatives (3 out of 7) of a single generation (of 3 alive generations) in this family. On pedigree analysis and sequencing of their mitochondrial DNA (mtDNA), a novel homoplasmic mutation of the mitochondrial tRNACys gene (5802A>G) was identified in these individuals. This mutation correlated with a destabilized conserved base pair in this tRNA anticodon stem. Position 30 is known to be crucial for carrying out effective codon recognition and stability of tRNA. In accordance with the importance of this conserved site, we observed that the predicted structure of tRNACys with the mutation was noticeably remodeled in a molecular dynamics simulation when compared with the isoform of the wild-type. All other 46 mutations observed in the individual's mtDNA were known variants belonging to haplogroup D4. Thus, this is the first report that provides evidence of the association between a mutation in tRNA and an enhanced risk of maternally transmissible obesity, offering more insights into obesity and its underlying nature.
Asunto(s)
ADN Mitocondrial/genética , Mutación , Obesidad/genética , ARN de Transferencia de Cisteína/genética , Aumento de Peso/genética , Adulto , Pueblo Asiatico/genética , Índice de Masa Corporal , Niño , China , Femenino , Predisposición Genética a la Enfermedad , Haplotipos , Herencia , Humanos , Masculino , Simulación de Dinámica Molecular , Obesidad/diagnóstico , Obesidad/etnología , Obesidad/fisiopatología , Linaje , Fenotipo , Aumento de Peso/etnologíaRESUMEN
In Bacillus subtilis, the tRNACys lacks an encoded CCA 3' end. To gain insight into the role of CCAase and RNases in tRNACys processing, several mutant strains were generated. Northern blot and RT-PCR results suggest that enzymes other than CCAase can participate in CCA addition at the 3' end of the immature tRNACys.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Bacillus subtilis/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Conformación de Ácido Nucleico , ARN Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismoRESUMEN
American foulbrood (AFB) caused by Paenibacillus larvae is the most destructive honeybee bacterial disease and its dissemination via commercial bee pollen is an important mechanism for the spread of this bacterium. Because Mexico imports bee pollen from several countries, we developed a tRNACys-PCR strategy and complemented that strategy with MALDI-TOF MS and amplicon-16S rRNA gene analysis to evaluate the presence of P. larvae in pollen samples. P. larvae was not detected when the tRNACys-PCR approach was applied to spore-forming bacterial colonies obtained from three different locations and this result was validated by bacterial identification via MALDI-TOF MS. The genera identified in the latter analysis were Bacillus (fourteen species) and Paenibacillus (six) species. However, amplicon-16S rRNA gene analysis for taxonomic composition revealed a low presence of Paenibacillaceae with 0.3 to 16.2% of relative abundance in the commercial pollen samples analyzed. Within this family, P. larvae accounted for 0.01% of the bacterial species present in one sample. Our results indicate that the tRNACys-PCR, combined with other molecular tools, will be a useful approach for identifying P. larvae in pollen samples and will assist in controlling the spread of the pathogen.
Asunto(s)
Abejas/microbiología , Paenibacillus larvae/genética , Polen/microbiología , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Animales , Bacillus/genética , Técnicas de Amplificación de Ácido Nucleico , Paenibacillus larvae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estados UnidosRESUMEN
In many organisms, the UGA stop codon is recoded to insert selenocysteine (Sec) into proteins. Sec incorporation in bacteria is directed by an mRNA element, known as the Sec-insertion sequence (SECIS), located downstream of the Sec codon. Unlike other aminoacyl-tRNAs, Sec-tRNASec is delivered to the ribosome by a dedicated elongation factor, SelB. We recently identified a series of tRNASec-like tRNA genes distributed across Bacteria that also encode a canonical tRNASec. These tRNAs contain sequence elements generally recognized by cysteinyl-tRNA synthetase (CysRS). While some of these tRNAs contain a UCA Sec anticodon, most have a GCA Cys anticodon. tRNASec with GCA anticodons are known to recode UGA codons. Here we investigate the clostridial Desulfotomaculum nigrificans tRNASec-like tRNACys, and show that this tRNA is acylated by CysRS, recognized by SelB, and capable of UGA recoding with Cys in Escherichia coli. We named this non-canonical group of tRNACys as 'tRNAReC' (Recoding with Cys). We performed a comprehensive survey of tRNAReC genes to establish their phylogenetic distribution, and found that, in a particular lineage of clostridial Pelotomaculum, the Cys identity elements of tRNAReC had mutated. This novel tRNA, which contains a UCA anticodon, is capable of Sec incorporation in E. coli, albeit with lower efficiency relative to Pelotomaculum tRNASec. We renamed this unusual tRNASec derived from tRNAReC as 'tRNAReU' (Recoding with Sec). Together, our results suggest that tRNAReC and tRNAReU may serve as safeguards in the production of selenoproteins and - to our knowledge - they provide the first example of programmed codon-anticodon mispairing in bacteria.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Proteínas Bacterianas/genética , Cisteína/metabolismo , Escherichia coli/genética , ARN de Transferencia de Cisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Anticodón/genética , Anticodón/metabolismo , Proteínas Bacterianas/metabolismo , Codón de Terminación/química , Codón de Terminación/metabolismo , Desulfotomaculum/genética , Desulfotomaculum/metabolismo , Escherichia coli/metabolismo , Código Genético , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Peptococcaceae/genética , Peptococcaceae/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia de Cisteína/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Selenoproteínas/biosíntesisRESUMEN
Selenium (Se) is an essential trace element for several organisms and is mostly present in proteins as L-selenocysteine (Sec or U). Sec is synthesized on its L-seryl-tRNASec to produce Sec-tRNASec molecules by a dedicated selenocysteine synthesis machinery and incorporated into selenoproteins at specified in-frame UGA codons. UGA-Sec insertion is signaled by an mRNA stem-loop structure called the SElenoCysteine Insertion Sequence (SECIS). tRNASec transcription regulation and folding have been described showing its importance to Sec biosynthesis. Here, we discuss structural aspects of Sec-tRNASec and its role in Sec biosynthesis as well as Sec incorporation into selenoproteins. Defects in the Sec biosynthesis or incorporation pathway have been correlated with pathological conditions.
Asunto(s)
ARN de Transferencia de Cisteína/genética , Selenocisteína/biosíntesis , Animales , Codón de Terminación/química , Codón de Terminación/genética , Codón de Terminación/metabolismo , Humanos , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/metabolismo , Selenocisteína/genéticaRESUMEN
Cysteine can be synthesized by tRNA-dependent mechanism using a two-step indirect pathway, where O-phosphoseryl-tRNA synthetase (SepRS) catalyzes the ligation of a mismatching O-phosphoserine (Sep) to tRNACys followed by the conversion of tRNA-bounded Sep into cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS). In ancestral methanogens, a third protein SepCysE forms a bridge between the two enzymes to create a ternary complex named the transsulfursome. By combination of X-ray crystallography, SAXS and EM, together with biochemical evidences, here we show that the three domains of SepCysE each bind SepRS, SepCysS, and tRNACys, respectively, which mediates the dynamic architecture of the transsulfursome and thus enables a global long-range channeling of tRNACys between SepRS and SepCysS distant active sites. This channeling mechanism could facilitate the consecutive reactions of the two-step indirect pathway of Cys-tRNACys synthesis (tRNA-dependent cysteine biosynthesis) to prevent challenge of translational fidelity, and may reflect the mechanism that cysteine was originally added into genetic code.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Arqueales/metabolismo , Cisteína/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Código Genético/genética , Methanocaldococcus/genética , Methanocaldococcus/metabolismo , Microscopía Electrónica , Modelos Moleculares , Mutación , Fosfoserina/química , Fosfoserina/metabolismo , Unión Proteica , Conformación Proteica , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/genética , Dispersión del Ángulo PequeñoRESUMEN
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
Kallmann syndrome (KS) is an inherited developmental disorder defined as the association of hypogonadotropic hypogonadism and anosmia or hyposmia. KS has been shown to be a genetically heterogeneous disease with different modes of inheritance. However, variants in any of the causative genes identified so far are only found in approximately one third of KS patients, thus indicating that other genes or pathways remain to be discovered. Here, we report a large Han Chinese family with inherited KS which harbors two novel variants, KAL1 c.146G>T (p.Cys49Phe) and mitochondrial tRNA(cys) (m.5800A>G). Although two variants can't exert obvious effects on the migration of GnRH neurons, they show the synergistic effect, which can account for the occurrence of the disorder in this family. Furthermore, the disturbance of the mitochondrial cysteinyl-tRNA pathway can significantly affect the migration of GnRH cells in vitro and in vivo by influencing the chemomigration function of anosmin-1. Our work highlights a new mode of inheritance underlay the genetic etiology of KS and provide valuable clues to understand the disease development.
Asunto(s)
ADN Mitocondrial/genética , Proteínas de la Matriz Extracelular/genética , Síndrome de Kallmann/patología , Proteínas del Tejido Nervioso/genética , ARN de Transferencia de Cisteína/genética , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Pueblo Asiatico/genética , Secuencia de Bases , Movimiento Celular , China , ADN Mitocondrial/química , Proteínas de la Matriz Extracelular/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Células HEK293 , Humanos , Síndrome de Kallmann/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Conformación de Ácido Nucleico , Linaje , Fenotipo , Mutación Puntual , Interferencia de ARN , ARN de Transferencia de Cisteína/metabolismo , Pez Cebra/metabolismoRESUMEN
In vitro-transcribed suppressor tRNAs are commonly used in site-specific fluorescence labeling for protein and ribosome-bound nascent chains (RNCs) studies. Here, we describe the production of nonorthogonal Bacillus subtilis tRNA(cys)(Amber) from Escherichia coli, a process that is superior to in vitro transcription in terms of yield, ease of manipulation, and tRNA stability. As cysteinyl-tRNA synthetase was previously shown to aminoacylate tRNA(cys)(Amber) with lower efficiency, multiple tRNA synthetase mutants were designed to optimize aminoacylation. Aminoacylated tRNA was conjugated to a fluorophore to produce BODIPY FL-cysteinyl-tRNA(cys)(Amber), which was used to generate ribosome-bound nascent chains of different lengths with the fluorophore incorporated at various predetermined sites. This tRNA tool may be beneficial in the site-specific labeling of full-length proteins as well as RNCs for biophysical and biological research.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Bacillus subtilis/genética , Escherichia coli/genética , ARN de Transferencia de Cisteína/biosíntesis , ARN de Transferencia de Cisteína/química , Aminoacil-ARNt Sintetasas/genética , Sistema Libre de Células , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Técnicas In Vitro , Modelos Moleculares , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Bacteriano/biosíntesis , ARN Bacteriano/química , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genética , Aminoacilación de ARN de TransferenciaRESUMEN
Although there are increasing reports showed a positive link between mitochondrial tRNACys G5821A mutation and mitochondrial diseases. However, its role remained controversial. In this paper, we took a comprehensive data analysis concerning this mutation and clinical diseases. Our data indicated that this mutation lacked an evolutionary conservation and did not get involved in the thermodynamic change of tRNACys gene. Therefore, based on these observations, we proposed that G5821A mutation is not deleterious mutation.
Asunto(s)
Genes Mitocondriales , Mutación Puntual , ARN de Transferencia de Cisteína/genética , Animales , Humanos , Mamíferos/genéticaRESUMEN
Mitochondrial tRNA genes are the hot spots for mutations associated with essential hypertension. We report here the clinical and molecular genetic characterization of two Han Chinese pedigrees with materially inherited essential hypertension. Clinical evaluation revealed the variable severity and age-at-onset of hypertension among matrilineal relatives. In particular, the age-at-onset of hypertension in the maternal kindred ranged from 36 years to 79 years. The sequence analysis of entire mitochondrial genome in two probands showed that two probands carried the identical homoplasmic tRNAMet/tRNAGlnA4401G and tRNACysG5821A mutations and distinct sets of polymorphisms belonging to East Asian haplogroup C. The A4401G mutation may affect the processing of the precursors of tRNAMet and tRNAGln , thereby altering the tRNA metabolism. The tRNACys G5821A mutation is located in the acceptor stem of tRNACys. This mutation may abol-ish the predicted G6-C67 pairing and consequently affect the structure and stability of mitochondrial tRNACys, thereby leading to mitochondrial dysfunction. Therefore, these data suggested that the tRNAMet/tRNAGlnA4401G and tRNACys G5821A mutations are likely associated with essential hypertension in these two Chinese pedigrees.
Asunto(s)
Pueblo Asiatico/etnología , Etnicidad/genética , Hipertensión/genética , Mitocondrias/genética , Mutación , Linaje , ARN de Transferencia Aminoácido-Específico/genética , Adulto , Anciano , Anciano de 80 o más Años , Pueblo Asiatico/genética , Secuencia de Bases , Niño , Femenino , Genotipo , Humanos , Hipertensión/etnología , Masculino , Persona de Mediana Edad , ARN de Transferencia de Cisteína/genética , ARN de Transferencia de Glutamina/genética , ARN de Transferencia de Metionina/genéticaRESUMEN
RNA processing is an essential pathway in the regulation of genetic expression in the cell. In this work, Bacillus subtilis was used to understand the effects of mercury on the mechanism of tRNA metabolism. The CVAAS (cold vapor atomic absorption spectroscopy) method revealed that from the addition of HgCl2 (0.75 µg ml(-1)) during the bacterial exponential phase, ca. 48% of the added mercury was taken up by the cells. This led to an immediate reduction in the rate of cell division. During this response, we observed accumulation of species shorter than mature tRNA(Cys) over a 10 h period. We did not observe this accumulation for another five tRNAs analyzed. tRNA processing is largely dependent on RNase R and PNPase in B. subtilis. Thus, when the exonuclease PNPase was absent, we found that the shorter tRNA(Cys) species increased and mature tRNA(Cys) decreased after mercury addition, but this proportion changed during the time analyzed. However, in the absence of RNase R and PNPase the accumulation of the shorter tRNA(Cys) was more pronounced and the mature form was not recovered. In the single rnr mutant strain the shorter tRNA(Cys) was not observed. All together, we provide in vivo evidence that PNPase and RNase R are indispensable in controlling tRNA(Cys) quality in the presence of mercury.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Cloruro de Mercurio/toxicidad , ARN Bacteriano/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Northern Blotting , Mutación/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/genéticaRESUMEN
Protein structure networks are constructed for the identification of long-range signaling pathways in cysteinyl tRNA synthetase (CysRS). Molecular dynamics simulation trajectory of CysRS-ligand complexes were used to determine conformational ensembles in order to gain insight into the allosteric signaling paths. Communication paths between the anticodon binding region and the aminoacylation region have been identified. Extensive interaction between the helix bundle domain and the anticodon binding domain, resulting in structural rigidity in the presence of tRNA, has been detected. Based on the predicted model, six residues along the communication paths have been examined by mutations (single and double) and shown to mediate a coordinated coupling between anticodon recognition and activation of amino acid at the active site. This study on CysRS clearly shows that specific key residues, which are involved in communication between distal sites in allosteric proteins but may be elusive in direct structure analysis, can be identified from dynamics of protein structure networks.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Anticodón/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , ARN Bacteriano/metabolismo , ARN de Transferencia de Cisteína/metabolismo , Regulación Alostérica/fisiología , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Anticodón/química , Anticodón/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Mutación , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/genética , ARN de Transferencia de Cisteína/química , ARN de Transferencia de Cisteína/genéticaRESUMEN
O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.
Asunto(s)
Escherichia coli/genética , Código Genético , Ingeniería Genética , Fosfoserina/metabolismo , Modificación Traduccional de las Proteínas , ARN de Transferencia Aminoácido-Específico/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Anticodón , Cloranfenicol/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Codón de Terminación , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Humanos , MAP Quinasa Quinasa 1/biosíntesis , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 1/genética , Factor Tu de Elongación Peptídica , Ingeniería de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia Aminoácido-Específico/genética , ARN de Transferencia de Cisteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Aminoacilación de ARN de TransferenciaRESUMEN
Of the two tRNA(Cys) (GCA) genes, trnC1-GCA and trnC2-GCA, previously identified in mitochondrial genome of sugar beet, the former is a native gene and probably a pseudo-copy, whereas the latter, of unknown origin, is transcribed into a tRNA [tRNA(Cys2) (GCA)]. In this study, the trnC2-GCA sequence was mined from various public databases. To evaluate whether or not the trnC2-GCA sequence is located in the mitochondrial genome, the relative copy number of its sequence to nuclear gene was assessed in a number of angiosperm species, using a quantitative real-time PCR assay. The trnC2-GCA sequence was found to exist sporadically in the mitochondrial genomes of a wide range of angiosperms. The mitochondrial tRNA(Cys2) (GCA) species from sugar beet (Beta vulgaris), spinach (Spinacea oleracea) and cucumber (Cucumis sativus) were found to be aminoacylated, indicating that they may participate in translation. We also identified a sugar beet nuclear gene that encodes cysteinyl-tRNA synthetase, which is dual-targeted to mitochondria and plastids, and may aminoacylate tRNA(Cys2) (GCA). What is of particular interest is that trnC1-GCA and trnC2-GCA co-exist in the mitochondrial genomes of eight diverse angiosperms, including spinach, and that the spinach tRNA(Cys1) (GCA) is also aminoacylated. Taken together, our observations lead us to surmise that trnC2-GCA may have been horizontally transferred to a common ancestor of eudicots, followed by co-existence and dual expression of trnC1-GCA and trnC2-GCA in mitochondria with occasional loss or inactivation of either trnC-GCA gene during evolution.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Aminoacilación/genética , Beta vulgaris/genética , Genoma Mitocondrial/genética , Magnoliopsida/genética , ARN de Transferencia de Cisteína/metabolismo , Aminoacil-ARNt Sintetasas/metabolismo , Beta vulgaris/enzimología , Beta vulgaris/metabolismo , Evolución Biológica , ADN Complementario/genética , ADN Mitocondrial/genética , ADN de Plantas/genética , Bases de Datos de Ácidos Nucleicos , Dosificación de Gen , Transferencia de Gen Horizontal , Magnoliopsida/enzimología , Magnoliopsida/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN de Planta/genética , ARN de Transferencia de Cisteína/genética , Análisis de Secuencia de ADNRESUMEN
We generated a conditional CCase mutant of Bacillus subtilis to explore the participation in vivo of the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNA(Cys), which lacks an encoded CCA 3' end. We observed that shorter tRNA(Cys) species, presumably lacking CCA, only accumulated when the inducible Pspac : cca was introduced into an rnr mutant strain, but not in combination with pnp. We sequenced the tRNA 3' ends produced in the various mutant tRNA(Cys) species to detect maturation and decay intermediates and observed that decay of the tRNA(Cys) occurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNA(Cys) at position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results provide in vivo evidence for the participation of the B. subtilis cca gene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase in B. subtilis participates in the quality control of tRNA.