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1.
Elife ; 132024 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-38393777

RESUMEN

A new in vitro system called Rec-Seq sheds light on how mRNA molecules compete for the machinery that translates their genetic sequence into proteins.


Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Ribosomas/metabolismo , ARN Mensajero/metabolismo
2.
Nat Struct Mol Biol ; 31(3): 455-464, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38287194

RESUMEN

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.


Asunto(s)
Factor 4F Eucariótico de Iniciación , Iniciación de la Cadena Peptídica Traduccional , Humanos , Factor 4F Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , Codón Iniciador/metabolismo , Ribosomas/metabolismo , ADN Helicasas/metabolismo , Biosíntesis de Proteínas , Factor 4A Eucariótico de Iniciación/química , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo
4.
J Biol Chem ; 298(10): 102368, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35963437

RESUMEN

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.


Asunto(s)
Factor 4A Eucariótico de Iniciación , Factor 4E Eucariótico de Iniciación , Humanos , Adenosina Trifosfato/metabolismo , Factor 4A Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/química , Factor 4F Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , Nucleótidos/química , Unión Proteica , ARN Mensajero/metabolismo
5.
Nature ; 607(7917): 185-190, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35732735

RESUMEN

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.


Asunto(s)
Factor 1 Eucariótico de Iniciación , Factores Eucarióticos de Iniciación , ARN de Transferencia de Metionina , Subunidades Ribosómicas , Microscopía por Crioelectrón , Factor 1 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/genética , Humanos , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Metionina/metabolismo , Subunidades Ribosómicas/química , Subunidades Ribosómicas/metabolismo , Imagen Individual de Molécula
6.
Science ; 369(6508): 1220-1227, 2020 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-32883864

RESUMEN

A key step in translational initiation is the recruitment of the 43S preinitiation complex by the cap-binding complex [eukaryotic initiation factor 4F (eIF4F)] at the 5' end of messenger RNA (mRNA) to form the 48S initiation complex (i.e., the 48S). The 48S then scans along the mRNA to locate a start codon. To understand the mechanisms involved, we used cryo-electron microscopy to determine the structure of a reconstituted human 48S The structure reveals insights into early events of translation initiation complex assembly, as well as how eIF4F interacts with subunits of eIF3 near the mRNA exit channel in the 43S The location of eIF4F is consistent with a slotting model of mRNA recruitment and suggests that downstream mRNA is unwound at least in part by being "pulled" through the 40S subunit during scanning.


Asunto(s)
Factor 3 de Iniciación Eucariótica/química , Factor 4F Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , Adenosina Trifosfato/química , Codón Iniciador , Microscopía por Crioelectrón , Humanos , Hidrólisis , ARN Mensajero/química
7.
Artículo en Inglés | MEDLINE | ID: mdl-29959192

RESUMEN

The eukaryotic translation pathway has been studied for more than four decades, but the molecular mechanisms that regulate each stage of the pathway are not completely defined. This is in part because we have very little understanding of the kinetic framework for the assembly and disassembly of pathway intermediates. Steps of the pathway are thought to occur in the subsecond to second time frame, but most assays to monitor these events require minutes to hours to complete. Understanding translational control in sufficient detail will therefore require the development of assays that can precisely monitor the kinetics of the translation pathway in real time. Here, we describe the translation pathway from the perspective of its kinetic parameters, discuss advances that are helping us move toward the goal of a rigorous kinetic understanding, and highlight some of the challenges that remain.


Asunto(s)
Eucariontes/metabolismo , Biosíntesis de Proteínas/fisiología , Factor 4F Eucariótico de Iniciación/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/genética
8.
Proc Natl Acad Sci U S A ; 114(24): 6304-6309, 2017 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-28559306

RESUMEN

In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Adenosina Trifosfato/metabolismo , Codón Iniciador/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Polarización de Fluorescencia , Humanos , Cinética , Modelos Biológicos , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/genética
9.
Elife ; 4: e07314, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25875391

RESUMEN

The general translation initiation factor eIF2 is a major translational control point. Multiple signaling pathways in the integrated stress response phosphorylate eIF2 serine-51, inhibiting nucleotide exchange by eIF2B. ISRIB, a potent drug-like small molecule, renders cells insensitive to eIF2α phosphorylation and enhances cognitive function in rodents by blocking long-term depression. ISRIB was identified in a phenotypic cell-based screen, and its mechanism of action remained unknown. We now report that ISRIB is an activator of eIF2B. Our reporter-based shRNA screen revealed an eIF2B requirement for ISRIB activity. Our results define ISRIB as a symmetric molecule, show ISRIB-mediated stabilization of activated eIF2B dimers, and suggest that eIF2B4 (δ-subunit) contributes to the ISRIB binding site. We also developed new ISRIB analogs, improving its EC50 to 600 pM in cell culture. By modulating eIF2B function, ISRIB promises to be an invaluable tool in proof-of-principle studies aiming to ameliorate cognitive defects resulting from neurodegenerative diseases.


Asunto(s)
Acetamidas/química , Ciclohexilaminas/química , Factor 2B Eucariótico de Iniciación/genética , Fármacos Neuroprotectores/química , Nootrópicos/química , Subunidades de Proteína/genética , Acetamidas/síntesis química , Acetamidas/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ciclohexilaminas/síntesis química , Ciclohexilaminas/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2B Eucariótico de Iniciación/metabolismo , Expresión Génica , Genes Reporteros , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Células K562 , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/farmacología , Nootrópicos/síntesis química , Nootrópicos/farmacología , Fosforilación , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Relación Estructura-Actividad , Tapsigargina/antagonistas & inhibidores , Tapsigargina/farmacología
10.
J Biol Chem ; 289(46): 31827-31836, 2014 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-25246524

RESUMEN

The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.


Asunto(s)
Factor 2 Eucariótico de Iniciación/química , Factor 3 de Iniciación Eucariótica/química , Guanosina Trifosfato/química , ARN de Transferencia de Metionina/química , Anisotropía , Células HeLa , Humanos , Cinética , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Ribosomas/química , Espectrometría de Fluorescencia , Termodinámica
11.
Nucleic Acids Res ; 40(2): 905-13, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-21940399

RESUMEN

The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to occur by way of a ternary complex (TC) comprising eIF2, GTP and Met-tRNA(i). We have generated from purified human proteins a stable multifactor complex (MFC) comprising eIF1, eIF2, eIF3 and eIF5, similar to the MFC reported in yeast and plants. A human MFC free of the ribosome also is detected in HeLa cells and rabbit reticulocytes, indicating that it exists in vivo. In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the same rate as the TC. However, MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting that MFC components may play a role in the release of eIF2-GDP from the ribosome following AUG recognition. Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly together with the TC. However, the MFC protein components also bind individually to 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor complexes. Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are identified, but which one predominates in vivo remains to be elucidated.


Asunto(s)
Factores Eucarióticos de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Aminoacil-ARN de Transferencia/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Adenosina Difosfato/metabolismo , Animales , Factor 2 Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Conejos , Reticulocitos/metabolismo
12.
J Proteome Res ; 10(10): 4613-23, 2011 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-21854064

RESUMEN

Eukaryotic translation requires a suite of proteins known as eukaryotic initiation factors (eIFs). These molecular effectors oversee the highly regulated initiation phase of translation. Essential to eukaryotic translation initiation is the protein eIF2, a heterotrimeric protein composed of the individually distinct subunits eIF2α, eIF2ß, and eIF2γ. The ternary complex, formed when eIF2 binds to GTP and Met-tRNA(i), is responsible for shuttling Met-tRNA(i) onto the awaiting 40S ribosome. As a necessary component for translation initiation, much attention has been given to the phosphorylation of eIF2α. Despite several previous investigations into eIF2 phosphorylation, most have centered on α- or ß-subunit phosphorylation and little is known regarding γ-subunit phosphorylation. Herein, we report eight sites of phosphorylation on the largest eIF2 subunit with seven novel phosphosite identifications via high resolution mass spectrometry. Of the eight sites identified, three are located in either the switch regions or nucleotide binding pocket domain. In addition, we have identified a possible kinase of eIF2, protein kinase C (PKC), which is capable of phosphorylating threonine 66 (thr-66) on the intact heterotrimer. These findings may shed new light on the regulation of ternary complex formation and alternate molecular effectors involved in this process prior to 80S ribosome formation and subsequent translation elongation and termination.


Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Proteína Quinasa C/metabolismo , Proteómica/métodos , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espectrometría de Masas/métodos , Modelos Biológicos , Fosforilación , Proteína Quinasa C/química , Estructura Terciaria de Proteína , Proteoma , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Treonina/química
13.
Artículo en Inglés | MEDLINE | ID: mdl-21636924

RESUMEN

The binding between two universally conserved translation initiation factors, eIF5B and eIF1A, is important in the initiation step of eukaryotic protein synthesis on the ribosome. Through this interaction, eIF1A assists in recruiting eIF5B to the initiating 40S subunit; eIF5B then encourages the joining of the 60S subunit to form an initiating 80S ribosome. Here, the expression, purification, crystallization and preliminary X-ray analyses of eIF5BΔN and the eIF5BΔN-eIF1AΔN complex from Saccharomyces cerevisiae are reported. The crystal of eIF5BΔN diffracted to 2.45 Šresolution and belonged to space group P4(1)2(1)2, with unit-cell parameters a = b = 130.0, c = 71.7 Å. The asymmetric unit was estimated to contain one molecule. The initial phase was obtained by Se-SAD. The crystal of the eIF5BΔN-eIF1AΔN complex diffracted to 3.3 Šresolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 101.9, b = 120.9, c = 132.8 Å. The asymmetric unit was estimated to contain two complex molecules.


Asunto(s)
Factor 1 Eucariótico de Iniciación/química , Factores Eucarióticos de Iniciación/química , Saccharomyces cerevisiae/química , Cristalización , Cristalografía por Rayos X , Factor 1 Eucariótico de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo
14.
Biochem J ; 433(1): 205-13, 2011 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-20942800

RESUMEN

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.


Asunto(s)
Acetiltransferasas/metabolismo , Lisina/análogos & derivados , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/metabolismo , Acetilación , Animales , Bovinos , Línea Celular Tumoral , Humanos , Lisina/metabolismo , Unión Proteica , Factor 5A Eucariótico de Iniciación de Traducción
15.
Protein Eng Des Sel ; 24(3): 301-9, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21131325

RESUMEN

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.


Asunto(s)
Escherichia coli/genética , Lisina/análogos & derivados , Oxigenasas de Función Mixta/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/genética , Animales , Bovinos , Clonación Molecular , Escherichia coli/citología , Expresión Génica , Vectores Genéticos/genética , Humanos , Oxigenasas de Función Mixta/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/biosíntesis , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Temperatura , Factores de Tiempo , Factor 5A Eucariótico de Iniciación de Traducción
16.
J Am Soc Mass Spectrom ; 20(9): 1699-706, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19564121

RESUMEN

The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins. Research presented herein is the first report showing retention of solution conformation of a protein as evidenced by CCS measurements of both recombinant and solvent disrupted versions of the same protein.


Asunto(s)
Espectrometría de Masas/métodos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/ultraestructura , Modelos Químicos , Solventes/química , Simulación por Computador , Conformación Proteica , Desnaturalización Proteica , Subunidades de Proteína
17.
Proc Natl Acad Sci U S A ; 106(27): 11028-33, 2009 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-19549823

RESUMEN

Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA(Ala) and hydrolysis of mis-acylated Ser- and Gly-tRNA(Ala) at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 A from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.


Asunto(s)
Alanina-ARNt Ligasa/química , Pyrococcus horikoshii/enzimología , Bacterias/enzimología , Biocatálisis , Dominio Catalítico , Activación Enzimática , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN de Transferencia/química
18.
Biochem Biophys Res Commun ; 371(4): 596-9, 2008 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-18381064

RESUMEN

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of alpha-, beta-, and gamma-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the alpha- and gamma-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The alpha-subunits bound to the gamma-subunit with large heat capacity change (DeltaC(p)) values. The DeltaH and DeltaC(p) values for the interaction between the alpha- and gamma-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the alpha-subunit and GTP changes the conformation of the switch region of the gamma-subunit and increases the affinity of the gamma-subunit for tRNA.


Asunto(s)
Proteínas Arqueales/química , Guanosina Trifosfato/química , Factor 2 Procariótico de Iniciación/química , Termodinámica , Proteínas Arqueales/genética , Cristalografía , Escherichia coli/genética , Guanosina Difosfato/química , Guanosina Trifosfato/análogos & derivados , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/aislamiento & purificación , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Sulfolobus solfataricus
19.
Proc Natl Acad Sci U S A ; 103(35): 13016-21, 2006 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-16924118

RESUMEN

Archaeal/eukaryotic initiation factor 2 (a/eIF2) consists of alpha-, beta-, and gamma-subunits and delivers initiator methionine tRNA (Met-tRNA(i)) to a small ribosomal subunit in a GTP-dependent manner. The structures of the aIF2betagamma (archaeal initiation factor 2 betagamma) heterodimeric complex in the apo and GDP forms were analyzed at 2.8- and 3.4-A resolution, respectively. The results showed that the N-terminal helix and the central helix-turn-helix domain of the beta-subunit bind to the G domain of the gamma-subunit but are distant from domains 2 and 3, to which the alpha-subunit and Met-tRNA(i) bind. This result is consistent with most of the previous analyses of eukaryotic factors, and thus indicates that the binding mode is essentially conserved among a/eIF2. Comparison with the uncomplexed structure showed significant differences between the two forms of the beta-subunit, particularly the C-terminal zinc-binding domain, which does not interact with the gamma-subunit and was suggested previously to be involved in GTP hydrolysis. Furthermore, the switch 1 region in the gamma-subunit, which is shown to be responsible for Met-tRNA(i) binding by mutational analysis, is moved away from the nucleotide through the interaction with highly conserved R87 in the beta-subunit. These results implicate that conformational change of the beta-subunit facilitates GTP hydrolysis by inducing the conformational change of the switch 1 region toward the off state.


Asunto(s)
Proteínas Arqueales/química , Guanosina Difosfato/química , Factores de Iniciación de Péptidos/química , Subunidades de Proteína/química , Pyrococcus furiosus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN de Transferencia de Metionina/química , Alineación de Secuencia
20.
Proc Natl Acad Sci U S A ; 102(33): 11669-74, 2005 Aug 16.
Artículo en Inglés | MEDLINE | ID: mdl-16087889

RESUMEN

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).


Asunto(s)
Alanina/genética , Alanina/metabolismo , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Edición de ARN , Acilación , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/genética , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Pyrococcus horikoshii/enzimología , ARN de Transferencia de Alanina/genética , ARN de Transferencia de Alanina/metabolismo , Alineación de Secuencia , Serina/genética , Serina/metabolismo , Especificidad por Sustrato , Treonina/genética , Treonina/metabolismo , Zinc/metabolismo
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