RESUMEN
The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.
Asunto(s)
Mycobacterium , Ribosomas , Ribosomas/genética , ARN de Transferencia/química , Mycobacterium/genéticaRESUMEN
In Fig. 1b of this Article, a U was inadvertently inserted after G15 in the D loop. The original Article has not been corrected.
RESUMEN
Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with -1 frameshifting.
Asunto(s)
Sistema de Lectura Ribosómico/genética , Extensión de la Cadena Peptídica de Translación/genética , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Ribosomas/metabolismo , Anticodón/genética , Sitios de Unión/genética , Proteínas Portadoras/genética , Codón/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , ARN Mensajero/genética , Sistemas de Lectura/genéticaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Enfermedad de Charcot-Marie-Tooth/etiología , Discapacidades del Desarrollo/etiología , Enfermedades del Cabello/etiología , Microcefalia/etiología , Mutación , Enfermedades de la Uña/etiología , Adulto , Secuencia de Aminoácidos , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/patología , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/patología , Femenino , Genes Recesivos , Predisposición Genética a la Enfermedad , Enfermedades del Cabello/enzimología , Enfermedades del Cabello/patología , Humanos , Masculino , Microcefalia/enzimología , Microcefalia/patología , Enfermedades de la Uña/enzimología , Enfermedades de la Uña/patología , Linaje , Fenotipo , Pronóstico , Homología de Secuencia , Adulto JovenRESUMEN
OBJECTIVE: The objective of this study was to identify the genetic cause for progressive peripheral nerve disease in a Venezuelan family. Despite the growing list of genes associated with Charcot-Marie-Tooth disease, many patients with axonal forms lack a genetic diagnosis. METHODS: A pedigree was constructed, based on family clinical data. Next-generation sequencing of mitochondrial DNA (mtDNA) was performed for 6 affected family members. Muscle biopsies from 4 family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Ultrastructural studies were performed on sensory nerve biopsies from 2 affected family members. RESULTS: Electrodiagnostic testing showed a motor and sensory axonal polyneuropathy. Pedigree analysis revealed inheritance only through the maternal line, consistent with mitochondrial transmission. Sequencing of mtDNA identified a mutation in the mitochondrial tRNAVal (mt-tRNAVal ) gene, m.1661A>G, present at nearly 100% heteroplasmy, which disrupts a Watson-Crick base pair in the T-stem-loop. Muscle biopsies showed chronic denervation/reinnervation changes, whereas biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple ETC complexes. Northern blots from skeletal muscle total RNA showed severe reduction in abundance of mt-tRNAVal , and mildly increased mt-tRNAPhe , in subjects compared with unrelated age- and sex-matched controls. Nerve biopsies from 2 affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy, and crystalline arrays) consistent with a mitochondrial neuropathy. CONCLUSION: We identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mt-tRNAVal , in a Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA gene. ANN NEUROL 2020;88:830-842.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , ARN Mitocondrial/genética , ARN de Transferencia/genética , Adolescente , Adulto , Anciano de 80 o más Años , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Linaje , Venezuela , Adulto JovenRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.
Asunto(s)
Alanina-ARNt Ligasa/genética , Aminoacil-ARNt Sintetasas/genética , Enfermedad de Charcot-Marie-Tooth/genética , ARN de Transferencia/genética , Adulto , Alelos , Aminoácidos/genética , Animales , Enfermedad de Charcot-Marie-Tooth/patología , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Heterogeneidad Genética , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Linaje , Levaduras/genética , Pez Cebra/genéticaRESUMEN
Amino acids are attached to the tRNA 3'-end as a prerequisite for entering the ribosome for protein synthesis. Amino acid attachment also gives tRNA access to nonribosomal cellular activities. However, the normal attachment is via an ester linkage between the carboxylic group of the amino acid and the 3'-hydroxyl of the terminal A76 ribose in tRNA. The instability of this ester linkage has severely hampered studies of aminoacyl-tRNAs. Although the use of 3'-amino-3'-deoxy A76 in a 3'-amino-tailed tRNA provides stable aminoacyl attachment via an amide linkage, there are multiple tailing protocols and the efficiency of each relative to the others is unknown. Here we compare five different tailing protocols in parallel, all dependent on the CCA-adding enzyme [CTP(ATP): tRNA nucleotidyl transferase; abbreviated as the CCA enzyme] to exchange the natural ribose with the modified one. We show that the most efficient protocol is achieved by the CCA-catalyzed pyrophosphorolysis removal of the natural A76 in equilibrium with the addition of the appropriate ATP analog to synthesize the modified 3'-end. This protocol for 3'-amino-tailing affords quantitative and stable attachment of a broad range of amino acids to tRNA, indicating its general utility for studies of aminoacyl-tRNAs in both canonical and noncanonical activities.
Asunto(s)
Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genética , ARN de Transferencia/genética , Adenosina Trifosfato/química , Aminoácidos/química , Aminoacil-ARNt Sintetasas/química , Sitios de Unión/genética , Catálisis , Escherichia coli/química , Escherichia coli/genética , Biosíntesis de Proteínas/genética , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genéticaRESUMEN
Ligation of tRNAs with their cognate amino acids, by aminoacyl-tRNA synthetases, establishes the genetic code. Throughout evolution, tRNA(Ala) selection by alanyl-tRNA synthetase (AlaRS) has depended predominantly on a single wobble base pair in the acceptor stem, G3â¢U70, mainly on the kcat level. Here we report the crystal structures of an archaeal AlaRS in complex with tRNA(Ala) with G3â¢U70 and its A3â¢U70 variant. AlaRS interacts with both the minor- and the major-groove sides of G3â¢U70, widening the major groove. The geometry difference between G3â¢U70 and A3â¢U70 is transmitted along the acceptor stem to the 3'-CCA region. Thus, the 3'-CCA region of tRNA(Ala) with G3â¢U70 is oriented to the reactive route that reaches the active site, whereas that of the A3â¢U70 variant is folded back into the non-reactive route. This novel mechanism enables the single wobble pair to dominantly determine the specificity of tRNA selection, by an approximate 100-fold difference in kcat.
Asunto(s)
Alanina-ARNt Ligasa/química , Archaeoglobus fulgidus/enzimología , Archaeoglobus fulgidus/genética , Emparejamiento Base , ARN de Transferencia de Alanina/química , ARN de Transferencia de Alanina/genética , Aminoacilación de ARN de Transferencia , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Especificidad por SustratoRESUMEN
Active tRNAs are extensively post-transcriptionally modified, particularly at the wobble position 34 and the position 37 on the 3'-side of the anticodon. The 5-carboxy-methoxy modification of U34 (cmo5U34) is present in Gram-negative tRNAs for six amino acids (Ala, Ser, Pro, Thr, Leu and Val), four of which (Ala, Ser, Pro and Thr) have a terminal methyl group to form 5-methoxy-carbonyl-methoxy-uridine (mcmo5U34) for higher reading-frame accuracy. The molecular basis for the selective terminal methylation is not understood. Many cmo5U34-tRNAs are essential for growth and cannot be substituted for mutational analysis. We show here that, with a novel genetic approach, we have created and isolated mutants of Escherichia coli tRNAPro and tRNAVal for analysis of the selective terminal methylation. We show that substitution of G35 in the anticodon of tRNAPro inactivates the terminal methylation, whereas introduction of G35 to tRNAVal confers it, indicating that G35 is a major determinant for the selectivity. We also show that, in tRNAPro, the terminal methylation at U34 is dependent on the primary m1G methylation at position 37 but not vice versa, indicating a hierarchical ranking of modifications between positions 34 and 37. We suggest that this hierarchy provides a mechanism to ensure top performance of a tRNA inside of cells.
Asunto(s)
Anticodón/genética , Conformación de Ácido Nucleico , ARN de Transferencia de Prolina/genética , ARN de Transferencia/genética , Secuencia de Bases , Codón/genética , Escherichia coli/genética , Metilación , ARN Bacteriano/genética , Uridina/análogos & derivados , Uridina/genéticaRESUMEN
Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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Aptámeros de Nucleótidos/química , Biosíntesis de Proteínas , Sondas ARN/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Aptámeros de Nucleótidos/metabolismo , Compuestos de Bencilo/química , Escherichia coli/genética , Colorantes Fluorescentes , Imidazolinas/química , Microscopía Fluorescente , Ribosomas/metabolismo , Spinacia oleracea/genéticaRESUMEN
In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Biosíntesis de Proteínas , Subunidades Ribosómicas/metabolismo , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Cinética , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Subunidades Ribosómicas/química , Subunidades Ribosómicas/genéticaRESUMEN
In Salmonella enterica serovar Typhimurium, Mg2+ limitation induces transcription of the mgtA Mg2+ transport gene, but the mechanism involved is unclear. The 5' leader of the mgtA mRNA contains a 17-codon, proline-rich ORF, mgtL, whose translation regulates the transcription of mgtA [Park S-Y et al. (2010) Cell 142:737-748]. Rapid translation of mgtL promotes formation of a secondary structure in the mgtA mRNA that permits termination of transcription by the Rho protein upstream of mgtA, whereas slow or incomplete translation of mgtL generates a different structure that blocks termination. We identified the following mutations that conferred high-level transcription of mgtA at high [Mg2+]: (i) a base-pair change that introduced an additional proline codon into mgtL, generating three consecutive proline codons; (ii) lesions in rpmA and rpmE, which encode ribosomal proteins L27 and L31, respectively; (iii) deletion of efp, which encodes elongation factor EF-P that assists the translation of proline codons; and (iv) a heat-sensitive mutation in trmD, whose product catalyzes the m1G37 methylation of tRNAPro Furthermore, substitution of three of the four proline codons in mgtL rendered mgtA uninducible. We hypothesize that the proline codons present an impediment to the translation of mgtL, which can be alleviated by high [Mg2+] exerted on component(s) of the translation machinery, such as EF-P, TrmD, or a ribosomal factor. Inadequate [Mg2+] precludes this alleviation, making mgtL translation inefficient and thereby permitting mgtA transcription. These findings are a significant step toward defining the target of Mg2+ in the regulation of mgtA transcription.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Magnesio/química , Proteínas de Transporte de Membrana/metabolismo , Péptidos/química , Prolina/química , Salmonella typhimurium/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Codón , Proteínas de Escherichia coli/química , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana/genética , Mutación , Factores de Elongación de Péptidos/química , Péptidos/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Biosíntesis de Proteínas , ARN de Transferencia/química , Ribosomas/química , Ribosomas/metabolismo , Transcripción Genética/efectos de los fármacos , ARNt Metiltransferasas/químicaRESUMEN
Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.
Asunto(s)
Anomalías Múltiples/genética , Alanina-ARNt Ligasa/genética , Epilepsia/genética , Modelos Moleculares , Vaina de Mielina/patología , Enfermedades del Sistema Nervioso Periférico/genética , Fenotipo , Anomalías Múltiples/patología , Alanina-ARNt Ligasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Epilepsia/patología , Genes Recesivos/genética , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Mutación/genética , Enfermedades del Sistema Nervioso Periférico/patología , Estudios Prospectivos , Análisis de Secuencia de ADN , Síndrome , Estados UnidosRESUMEN
The deep trefoil knot architecture is unique to the SpoU and tRNA methyltransferase D (TrmD) (SPOUT) family of methyltransferases (MTases) in all three domains of life. In bacteria, TrmD catalyzes the N(1)-methylguanosine (m(1)G) modification at position 37 in transfer RNAs (tRNAs) with the (36)GG(37) sequence, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. The m(1)G37-modified tRNA functions properly to prevent +1 frameshift errors on the ribosome. Here we report the crystal structure of the TrmD homodimer in complex with a substrate tRNA and an AdoMet analog. Our structural analysis revealed the mechanism by which TrmD binds the substrate tRNA in an AdoMet-dependent manner. The trefoil-knot center, which is structurally conserved among SPOUT MTases, accommodates the adenosine moiety of AdoMet by loosening/retightening of the knot. The TrmD-specific regions surrounding the trefoil knot recognize the methionine moiety of AdoMet, and thereby establish the entire TrmD structure for global interactions with tRNA and sequential and specific accommodations of G37 and G36, resulting in the synthesis of m(1)G37-tRNA.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Haemophilus influenzae/enzimología , ARN de Transferencia/metabolismo , Thermotoga maritima/enzimología , ARNt Metiltransferasas/química , ARNt Metiltransferasas/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Secuencia de Aminoácidos , Anticodón/genética , Secuencia de Bases , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Guanina/metabolismo , Cinética , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , ARN de Transferencia/química , ARN de Transferencia/genética , S-Adenosilmetionina , Alineación de Secuencia , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bifunctional ARS that charges tRNAGly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here, we describe a patient from the NIH Undiagnosed Diseases Program with a multisystem, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Discapacidades del Desarrollo/genética , Genes Recesivos , Glicina-ARNt Ligasa/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico por imagen , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Niño , Citoplasma/enzimología , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/fisiopatología , Femenino , Variación Genética , Humanos , Mitocondrias/enzimología , Secuenciación del ExomaRESUMEN
The intrinsic apoptosis pathway occurs through the release of mitochondrial cytochrome c to the cytosol, where it promotes activation of the caspase family of proteases. The observation that tRNA binds to cytochrome c revealed a previously unexpected mode of apoptotic regulation. However, the molecular characteristics of this interaction, and its impact on each interaction partner, are not well understood. Using a novel fluorescence assay, we show here that cytochrome c binds to tRNA with an affinity comparable with other tRNA-protein binding interactions and with a molecular ratio of â¼3:1. Cytochrome c recognizes the tertiary structural features of tRNA, particularly in the core region. This binding is independent of the charging state of tRNA but is regulated by the redox state of cytochrome c. Compared with reduced cytochrome c, oxidized cytochrome c binds to tRNA with a weaker affinity, which correlates with its stronger pro-apoptotic activity. tRNA binding both facilitates cytochrome c reduction and inhibits the peroxidase activity of cytochrome c, which is involved in its release from mitochondria. Together, these findings provide new insights into the cytochrome c-tRNA interaction and apoptotic regulation.
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Citocromos c/química , ARN de Transferencia/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Animales , Apoptosis/fisiología , Bovinos , Citocromos c/genética , Citocromos c/metabolismo , Humanos , Unión Proteica , Estructura Cuaternaria de Proteína , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: While the CCA sequence at the mature 3' end of tRNAs is conserved and critical for translational function, a genetic template for this sequence is not always contained in tRNA genes. In eukaryotes and Archaea, the CCA ends of tRNAs are synthesized post-transcriptionally by CCA-adding enzymes. In Bacteria, tRNA genes template CCA sporadically. RESULTS: In order to understand the variation in how prokaryotic tRNA genes template CCA, we re-annotated tRNA genes in tRNAdb-CE database version 0.8. Among 132,129 prokaryotic tRNA genes, initiator tRNA genes template CCA at the highest average frequency (74.1%) over all functional classes except selenocysteine and pyrrolysine tRNA genes (88.1% and 100% respectively). Across bacterial phyla and a wide range of genome sizes, many lineages exist in which predominantly initiator tRNA genes template CCA. Convergent and parallel retention of CCA templating in initiator tRNA genes evolved in independent histories of reductive genome evolution in Bacteria. Also, in a majority of cyanobacterial and actinobacterial genera, predominantly initiator tRNA genes template CCA. We also found that a surprising fraction of archaeal tRNA genes template CCA. CONCLUSIONS: We suggest that cotranscriptional synthesis of initiator tRNA CCA 3' ends can complement inefficient processing of initiator tRNA precursors, "bootstrap" rapid initiation of protein synthesis from a non-growing state, or contribute to an increase in cellular growth rates by reducing overheads of mass and energy to maintain nonfunctional tRNA precursor pools. More generally, CCA templating in structurally non-conforming tRNA genes can afford cells robustness and greater plasticity to respond rapidly to environmental changes and stimuli.
Asunto(s)
Bacterias/genética , Precursores del ARN/metabolismo , Anticodón , Archaea/genética , Emparejamiento Base , Secuencia de Bases , Bases de Datos Genéticas , Genes Arqueales , Genes Bacterianos , Precursores del ARN/química , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismoRESUMEN
Aminoacyl-tRNAs are the biologically active substrates for peptide bond formation in protein synthesis. The stability of the acyl linkage in each aminoacyl-tRNA, formed through an ester bond that connects the amino acid carboxyl group with the tRNA terminal 3'-OH group, is thus important. While the ester linkage is the same for all aminoacyl-tRNAs, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. Here, we show, by analysis of the half-lives of 12 of the 22 natural aminoacyl-tRNAs used in peptide bond formation, that the stability of the acyl linkage is effectively determined only by the chemical nature of the amino acid side chain. Even the chirality of the side chain exhibits little influence. Proline confers the lowest stability to the linkage, while isoleucine and valine confer the highest, whereas the nucleotide sequence in the tRNA provides negligible contribution to the stability. We find that, among the variables tested, the protein translation factor EF-Tu is the only one that can protect a weak acyl linkage from hydrolysis. These results suggest that each amino acid plays an active role in determining its own stability in the acyl linkage to tRNA, but that EF-Tu overrides this individuality and protects the acyl linkage stability for protein synthesis on the ribosome.
Asunto(s)
Aminoácidos/genética , Aminoácidos/metabolismo , Biosíntesis de Proteínas/genética , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Semivida , Hidrólisis , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismoRESUMEN
We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.