Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Nat Commun ; 15(1): 4094, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38750017

RESUMEN

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.


Asunto(s)
Microscopía por Crioelectrón , ARN de Transferencia , Humanos , ARN de Transferencia/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , Uridina/química , Uridina/metabolismo , Mutación , Acetilcoenzima A/metabolismo , Acetilcoenzima A/química , Modelos Moleculares , Acetilación , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Unión Proteica
2.
Sci Adv ; 9(2): eadd9688, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36638176

RESUMEN

Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.


Asunto(s)
Citocromos b , Plastocianina , Citocromos b/metabolismo , Plastocianina/metabolismo , Microscopía por Crioelectrón , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/metabolismo , Oxidación-Reducción , Fotosíntesis , Plantas/metabolismo , Quinonas , Transporte de Electrón
3.
Nucleic Acids Res ; 51(5): 2011-2032, 2023 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-36617428

RESUMEN

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.


The multi-subunit Elongator complex mediates the addition of a carboxymethyl group to wobble uridines in eukaryotic tRNAs. This tRNA modification is crucial to preserve the integrity of cellular proteomes and to protects us against severe neurodegenerative diseases. Elongator is organized in two distinct modules (i) the larger Elp123 subcomplex that binds and modifies the suitable tRNA substrate and (ii) the smaller Elp456 subcomplex that assists the release of the modified tRNA. The presented cryo-EM structures of Elongator show that the assemblies are very dynamic and undergo conformational rearrangements at consecutive steps of the process. Last but not least, the study provides a detailed reaction scheme and shows that the architecture of Elongator is highly conserved from yeast to mammals.


Asunto(s)
Complejos Multiproteicos , Extensión de la Cadena Peptídica de Translación , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Animales , Humanos , Ratones , Microscopía por Crioelectrón , Histona Acetiltransferasas/metabolismo , Unión Proteica , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura
4.
DNA Repair (Amst) ; 85: 102746, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31739207

RESUMEN

Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its ß-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway.


Asunto(s)
Bacterias/genética , ADN Helicasas/química , ADN Helicasas/metabolismo , Adenosina Trifosfatasas/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Modelos Moleculares , Conformación Proteica
5.
Sci Adv ; 5(7): eaaw2326, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31309145

RESUMEN

The highly conserved Elongator complex modifies transfer RNAs (tRNAs) in their wobble base position, thereby regulating protein synthesis and ensuring proteome stability. The precise mechanisms of tRNA recognition and its modification reaction remain elusive. Here, we show cryo-electron microscopy structures of the catalytic subcomplex of Elongator and its tRNA-bound state at resolutions of 3.3 and 4.4 Å. The structures resolve details of the catalytic site, including the substrate tRNA, the iron-sulfur cluster, and a SAM molecule, which are all validated by mutational analyses in vitro and in vivo. tRNA binding induces conformational rearrangements, which precisely position the targeted anticodon base in the active site. Our results provide the molecular basis for substrate recognition of Elongator, essential to understand its cellular function and role in neurodegenerative diseases and cancer.


Asunto(s)
Complejos Multiproteicos/metabolismo , Factores de Elongación de Péptidos/metabolismo , ARN de Transferencia/genética , Anticodón/química , Sitios de Unión , Dominio Catalítico , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Modelos Moleculares , Conformación Molecular , Complejos Multiproteicos/química , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Unión Proteica , ARN de Transferencia/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
FEBS Lett ; 592(4): 502-515, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28960290

RESUMEN

Nucleoside modifications in tRNA anticodons regulate ribosome dynamics during translation elongation and, thereby, fine-tune global protein synthesis rates. The highly conserved eukaryotic Elongator complex conducts specific C5-substitutions in tRNA wobble base uridines. It harbors two copies of each of its six individual subunits, which are all equally important for its activity. Here, we summarize recent developments focusing on the architecture of the Elongator complex, showing an asymmetric subunit arrangement, and its functional implications. In addition, we discuss the role of its proposed active site, its individual subunits and temporarily associated regulatory factors. Finally, we aim to provide mechanistic explanations for the link between mutations in Elongator subunits and the onset of several severe human pathologies.


Asunto(s)
Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Animales , Humanos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , ARN de Transferencia/genética , ARN de Transferencia/metabolismo
7.
Nat Commun ; 7: 12568, 2016 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-27562541

RESUMEN

Nucleotide excision repair (NER) removes chemically diverse DNA lesions in all domains of life. In Escherichia coli, UvrA and UvrB initiate NER, although the mechanistic details of how this occurs in vivo remain to be established. Here, we use single-molecule fluorescence imaging to provide a comprehensive characterization of the lesion search, recognition and verification process in living cells. We show that NER initiation involves a two-step mechanism in which UvrA scans the genome and locates DNA damage independently of UvrB. Then UvrA recruits UvrB from solution to the lesion. These steps are coordinated by ATP binding and hydrolysis in the 'proximal' and 'distal' UvrA ATP-binding sites. We show that initial UvrB-independent damage recognition by UvrA requires ATPase activity in the distal site only. Subsequent UvrB recruitment requires ATP hydrolysis in the proximal site. Finally, UvrA dissociates from the lesion complex, allowing UvrB to orchestrate the downstream NER reactions.


Asunto(s)
Adenosina Trifosfatasas/fisiología , ADN Helicasas/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Adenosina Trifosfato/metabolismo , Daño del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Hidrólisis , Microscopía/métodos , Modelos Moleculares , Unión Proteica/fisiología , Imagen Individual de Molécula/métodos
8.
Nucleic Acids Res ; 42(16): 10762-75, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25120270

RESUMEN

Rad2/XPG belongs to the flap nuclease family and is responsible for a key step of the eukaryotic nucleotide excision DNA repair (NER) pathway. To elucidate the mechanism of DNA binding by Rad2/XPG, we solved crystal structures of the catalytic core of Rad2 in complex with a substrate. Rad2 utilizes three structural modules for recognition of the double-stranded portion of DNA substrate, particularly a Rad2-specific α-helix for binding the cleaved strand. The protein does not specifically recognize the single-stranded portion of the nucleic acid. Our data suggest that in contrast to related enzymes (FEN1 and EXO1), the Rad2 active site may be more accessible, which would create an exit route for substrates without a free 5' end.


Asunto(s)
Proteínas de Unión al ADN/química , Endodesoxirribonucleasas/química , Proteínas de Saccharomyces cerevisiae/química , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Exodesoxirribonucleasas/química , Endonucleasas de ADN Solapado/química , Modelos Moleculares , Mutación , Proteínas Nucleares/genética , Unión Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética
9.
Nat Struct Mol Biol ; 18(2): 191-7, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21240268

RESUMEN

One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Thermotoga maritima/metabolismo , Adenosina Difosfato/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , ADN/química , Daño del ADN , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Thermotoga maritima/química
10.
J Neurochem ; 113(4): 848-59, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20180829

RESUMEN

The present study uses a proteomic approach to examine possible alterations of protein expression in the hippocampus of rats that are subjected to chronic mild stress (CMS). These rats serve as an animal model that was developed to mimic anhedonia, which is one of the core symptoms of depression. As antidepressant treatment is effective after a few weeks of administration, we also aimed to identify changes that were linked to chronic (once daily for 4 weeks) and 'pulse' (once a week) administration of imipramine. Fifteen differential proteins were identified with 2D electrophoresis followed by mass spectrometry. Although both methods of imipramine administration restored normal sucrose consumption in rats that were subjected to CMS, the molecular mechanisms of these two therapies were different. CMS-induced changes in the levels of dynactin 2, Ash 2, non-neuronal SNAP25 and alpha-enolase were reversed by chronic imipramine, but 'pulse' treatment was not that effective.


Asunto(s)
Giro Dentado/metabolismo , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/metabolismo , Imipramina/farmacología , Proteoma/metabolismo , Estrés Psicológico/metabolismo , Animales , Antidepresivos Tricíclicos/farmacología , Antidepresivos Tricíclicos/uso terapéutico , Apetito/efectos de los fármacos , Apetito/fisiología , Enfermedad Crónica , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Giro Dentado/fisiopatología , Trastorno Depresivo/etiología , Modelos Animales de Enfermedad , Complejo Dinactina , Electroforesis en Gel Bidimensional , Conducta Alimentaria/efectos de los fármacos , Conducta Alimentaria/fisiología , Imipramina/uso terapéutico , Masculino , Espectrometría de Masas , Proteínas Asociadas a Microtúbulos/efectos de los fármacos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/metabolismo , Fosfopiruvato Hidratasa/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Proteoma/efectos de los fármacos , Ratas , Ratas Wistar , Estrés Psicológico/complicaciones , Estrés Psicológico/fisiopatología , Proteína 25 Asociada a Sinaptosomas/efectos de los fármacos , Proteína 25 Asociada a Sinaptosomas/metabolismo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA