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1.
Int J Mol Sci ; 22(24)2021 Dec 08.
Artículo en Inglés | MEDLINE | ID: mdl-34948034

RESUMEN

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.


Asunto(s)
Escherichia coli/crecimiento & desarrollo , GTP Fosfohidrolasas/metabolismo , Genes Letales , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/metabolismo , ARN de Transferencia de Metionina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Trifosfato/química , Hidrólisis , Modelos Moleculares , Fenotipo , Factor 2 Procariótico de Iniciación/genética , Conformación Proteica , Dominios Proteicos , Ribosomas/química , Ribosomas/metabolismo
2.
Int J Mol Sci ; 21(3)2020 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-31979156

RESUMEN

The conserved Histidine 301 in switch II of Geobacillus stearothermophilus IF2 G2 domain was substituted with Ser, Gln, Arg, Leu and Tyr to generate mutants displaying different phenotypes. Overexpression of IF2H301S, IF2H301L and IF2H301Y in cells expressing wtIF2, unlike IF2H301Q and IF2H301R, caused a dominant lethal phenotype, inhibiting in vivo translation and drastically reducing cell viability. All mutants bound GTP but, except for IF2H301Q, were inactive in ribosome-dependent GTPase for different reasons. All mutants promoted 30S initiation complex (30S IC) formation with wild type (wt) efficiency but upon 30S IC association with the 50S subunit, the fMet-tRNA reacted with puromycin to different extents depending upon the IF2 mutant present in the complex (wtIF2 to IF2H301Q > IF2H301R >>> IF2H301S, IF2H301L and IF2H301Y) whereas only fMet-tRNA 30S-bound with IF2H301Q retained some ability to form initiation dipeptide fMet-Phe. Unlike wtIF2, all mutants, regardless of their ability to hydrolyze GTP, displayed higher affinity for the ribosome and failed to dissociate from the ribosomes upon 50S docking to 30S IC. We conclude that different amino acids substitutions of His301 cause different structural alterations of the factor, resulting in disparate phenotypes with no direct correlation existing between GTPase inactivation and IF2 failure to dissociate from ribosomes.


Asunto(s)
Proteínas Bacterianas/genética , Geobacillus stearothermophilus/genética , Histidina/genética , Mutación/genética , Factores de Iniciación de Péptidos/genética , Sustitución de Aminoácidos/genética , GTP Fosfohidrolasas/genética , Guanosina Trifosfato/genética , Fenotipo , Biosíntesis de Proteínas/genética , Dominios Proteicos/genética , ARN de Transferencia de Metionina/genética , Ribosomas/genética
3.
Nucleic Acids Res ; 47(9): 4638-4651, 2019 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-30916329

RESUMEN

After a 37 to 10°C temperature downshift the level of translation initiation factor IF2, like that of IF1 and IF3, increases at least 3-fold with respect to the ribosomes. To clarify the mechanisms and conditions leading to cold-stress induction of infB expression, the consequences of this temperature shift on infB (IF2) transcription, infB mRNA stability and translation were analysed. The Escherichia coli gene encoding IF2 is part of the metY-nusA-infB operon that contains three known promoters (P-1, P0 and P2) in addition to two promoters P3 and P4 identified in this study, the latter committed to the synthesis of a monocistronic mRNA encoding exclusively IF2. The results obtained indicate that the increased level of IF2 following cold stress depends on three mechanisms: (i) activation of all the promoters of the operon, P-1 being the most cold-responsive, as a likely consequence of the reduction of the ppGpp level that follows cold stress; (ii) a large increase in infB mRNA half-life and (iii) the cold-shock induced translational bias that ensures efficient translation of infB mRNA by the translational apparatus of cold shocked cells. A comparison of the mechanisms responsible for the cold shock induction of the three initiation factors is also presented.


Asunto(s)
Respuesta al Choque por Frío/genética , Factor 2 Procariótico de Iniciación/genética , Regiones Promotoras Genéticas/genética , Transcripción Genética , Frío , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Biosíntesis de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Ribosomas/genética
4.
Nucleic Acids Res ; 45(12): 7309-7325, 2017 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-28575317

RESUMEN

To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium. The behaviors of AUAmRNA, UUGmRNA and AUGmRNA were the same between 20 and 40°C whereas the GUG and AUUmRNAs were less active and competed poorly with the AUGmRNA, especially at low temperature. Nutrient limitation and preferential inhibition by ppGpp severely affected activity and competitiveness of all mRNAs bearing non-AUG starts, the UUGmRNA being the least affected. Overall, our data indicate that beyond these effects exclusively due to the degenerate start codons within an optimized translational initiation region, an important role is played by the context in which the rare start codons are present.


Asunto(s)
Codón Iniciador , Escherichia coli/genética , Iniciación de la Cadena Peptídica Traduccional , Factor 1 Procariótico de Iniciación/genética , ARN Mensajero/genética , Unión Competitiva , Escherichia coli/química , Escherichia coli/metabolismo , Cinética , Mutación , Factor 1 Procariótico de Iniciación/metabolismo , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Fracciones Subcelulares/química , Temperatura
5.
J Mol Biol ; 428(8): 1558-73, 2016 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-26953262

RESUMEN

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°â†’10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.


Asunto(s)
Escherichia coli/química , ARN Bacteriano/química , ARN Ribosómico 16S/química , ARN Ribosómico 23S/química , Aclimatación , Frío , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica , Operón , Fosfatos/química , Polirribosomas/química , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Subunidades Ribosómicas/química , Ribosomas/química , Temperatura , Factores de Tiempo , Transcripción Genética
6.
Cell Mol Life Sci ; 72(22): 4341-67, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26259514

RESUMEN

Initiation of mRNA translation is a major checkpoint for regulating level and fidelity of protein synthesis. Being rate limiting in protein synthesis, translation initiation also represents the target of many post-transcriptional mechanisms regulating gene expression. The process begins with the formation of an unstable 30S pre-initiation complex (30S pre-IC) containing initiation factors (IFs) IF1, IF2 and IF3, the translation initiation region of an mRNA and initiator fMet-tRNA whose codon and anticodon pair in the P-site following a first-order rearrangement of the 30S pre-IC produces a locked 30S initiation complex (30SIC); this is docked by the 50S subunit to form a 70S complex that, following several conformational changes, positional readjustments of its ligands and ejection of the IFs, becomes a 70S initiation complex productive in initiation dipeptide formation. The first EF-G-dependent translocation marks the beginning of the elongation phase of translation. Here, we review structural, mechanistic and dynamical aspects of this process.


Asunto(s)
Bacterias/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia de Metionina/genética , Bacterias/metabolismo , Sitios de Unión/genética , Codón Iniciador/genética , Codón Iniciador/metabolismo , Modelos Genéticos , Conformación de Ácido Nucleico , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo , Ribosomas/metabolismo
7.
Microbiologyopen ; 2(2): 293-307, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23420694

RESUMEN

Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock.


Asunto(s)
Proteínas Bacterianas/metabolismo , Respuesta al Choque por Frío/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Adaptación Fisiológica , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/fisiología , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Ligandos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Análisis de Secuencia de ADN , Temperatura
8.
Nucleic Acids Res ; 40(20): 10366-74, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-22941660

RESUMEN

Furvina®, also denominated G1 (MW 297), is a synthetic nitrovinylfuran [2-bromo-5-(2-bromo-2-nitrovinyl)-furan] antibiotic with a broad antimicrobial spectrum. An ointment (Dermofural®) containing G1 as the only active principle is currently marketed in Cuba and successfully used to treat dermatological infections. Here we describe the molecular target and mechanism of action of G1 in bacteria and demonstrate that in vivo G1 preferentially inhibits protein synthesis over RNA, DNA and cell wall synthesis. Furthermore, we demonstrate that G1 targets the small ribosomal subunit, binds at or near the P-decoding site and inhibits its function interfering with the ribosomal binding of fMet-tRNA during 30S initiation complex (IC) formation ultimately inhibiting translation. Notably, this G1 inhibition displays a bias for the nature (purine vs. pyrimidine) of the 3'-base of the codon, occurring efficiently only when the mRNA directing 30S IC formation and translation contains the canonical AUG initiation triplet or the rarely found AUA triplet, but hardly occurs when the mRNA start codon is either one of the non-canonical triplets AUU or AUC. This codon discrimination by G1 is reminiscent, though of opposite type of that displayed by IF3 in its fidelity function, and remarkably does not occur in the absence of this factor.


Asunto(s)
Antibacterianos/farmacología , Codón Iniciador , Furanos/farmacología , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Subunidades Ribosómicas Pequeñas Bacterianas/efectos de los fármacos , Compuestos de Vinilo/farmacología , Sitios de Unión , Subunidades Ribosómicas Pequeñas Bacterianas/química
9.
Nucleic Acids Res ; 40(16): 7946-55, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22723375

RESUMEN

Translation initiation factor IF2 is a guanine nucleotide-binding protein. The free energy change associated with guanosine triphosphate hydrolase (GTPase) activity of these proteins is believed to be the driving force allowing them to perform their functions as molecular switches. We examined role and relevance of IF2 GTPase and demonstrate that an Escherichia coli IF2 mutant bearing a single amino acid substitution (E571K) in its 30S binding domain (IF2-G3) can perform in vitro all individual translation initiation functions of wild type (wt) IF2 and supports faithful messenger RNA translation, despite having a reduced affinity for the 30S subunit and being completely inactive in GTP hydrolysis. Furthermore, the corresponding GTPase-null mutant of Bacillus stearothermophilus (E424K) can replace in vivo wt IF2 allowing an E. coli infB null mutant to grow with almost wt duplication times. Following the E571K (and E424K) mutation, which likely disrupts hydrogen bonding between subdomains G2 and G3, IF2 acquires a guanosine diphosphate (GDP)-like conformation, no longer responsive to GTP binding thereby highlighting the importance of interdomain communication in IF2. Our data underlie the importance of GTP as an IF2 ligand in the early initiation steps and the dispensability of the free energy generated by the IF2 GTPase in the late events of the translation initiation pathway.


Asunto(s)
GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Factor 2 Procariótico de Iniciación/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , GTP Fosfohidrolasas/genética , Geobacillus stearothermophilus/genética , Hidrólisis , Mutación , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/genética , Estructura Terciaria de Proteína , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo
10.
Gene ; 492(2): 382-8, 2012 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-22093878

RESUMEN

CspA is a small (7.4 kDa) nucleic acid binding protein of Escherichia coli whose expression is stimulated after cold-stress but whose level is also extraordinarily high during the early phase of growth of non-stressed cells. In this study the relationship existing between cspA transcription/translation on the one hand and the acquisition of critical mass for cell division and chromosome replication, on the other, in stationary phase cells subjected to a nutritional up-shift at 37 °C has been analyzed. Measurements of optical density and viable counts, pulse-chase, real-time PCR and immunodetection experiments, as well as cytofluorimetric and DNA duplication analyses show that synthesis of new CspA molecules at 37 °C is not only restricted to the lag phase ensuing the nutritional up-shift, but continues also during the first stages of logarithmic growth, when cells have already started dividing; although the early synthesized molecules are diluted by the following cell divisions and new synthesis occurs at an extremely low level, cspA mRNA and CspA continue to be present. A possible explanation for the apparent paradox that cspA is activated not only following cold stress, but also under non-stress and other stress conditions which entail a down-regulation of bulk gene expression and protein synthesis is presented.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Proteínas de Choque Térmico/metabolismo , Calor , División Celular , Proteínas y Péptidos de Choque por Frío , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Estabilidad Proteica
11.
Mol Cell ; 37(1): 21-33, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20129052

RESUMEN

Cold induction of cspA, the paradigm Escherichia coli cold-shock gene, is mainly subject to posttranscriptional control, partly promoted by cis-acting elements of its transcript, whose secondary structure at 37 degrees C and at cold-shock temperature has been elucidated here by enzymatic and chemical probing. The structures, which were also validated by mutagenesis, demonstrate that cspA mRNA undergoes a temperature-dependent structural rearrangement, likely resulting from stabilization in the cold of an otherwise thermodynamically unstable folding intermediate. At low temperature, the "cold-shock" structure is more efficiently translated and somewhat less susceptible to degradation than the 37 degrees C structure. Overall, our data shed light on a molecular mechanism at the basis of the cold-shock response, indicating that cspA mRNA is able to sense temperature downshifts, adopting functionally distinct structures at different temperatures, even without the aid of trans-acting factors. Unlike with other previously studied RNA thermometers, these structural rearrangements do not result from melting of hairpin structures.


Asunto(s)
Frío , Proteínas de Escherichia coli/fisiología , Escherichia coli/genética , Proteínas de Choque Térmico/fisiología , Biosíntesis de Proteínas , ARN Mensajero/fisiología , Regiones no Traducidas 5' , Aclimatación , Proteínas y Péptidos de Choque por Frío , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Modelos Genéticos , Conformación de Ácido Nucleico , ARN Mensajero/química
12.
J Mol Biol ; 396(1): 118-29, 2010 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19917289

RESUMEN

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive "cross-talking" between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.


Asunto(s)
Dominio Catalítico , Geobacillus stearothermophilus/metabolismo , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/metabolismo , Ribosomas/metabolismo , Sustitución de Aminoácidos/genética , Dipéptidos/metabolismo , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Prueba de Complementación Genética , Geobacillus stearothermophilus/genética , Cinética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Factor 2 Procariótico de Iniciación/genética , Unión Proteica , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Subunidades Ribosómicas/metabolismo , Eliminación de Secuencia
13.
J Biol Chem ; 284(44): 30453-62, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19740756

RESUMEN

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencias de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Espectroscopía de Resonancia Magnética , Regiones Promotoras Genéticas , Multimerización de Proteína , Proteínas Represoras
14.
Gene ; 428(1-2): 31-5, 2009 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-18951960

RESUMEN

Bacillus stearothermophilus infA encoding translation initiation factor IF1 was cloned and expressed in Escherichia coli and its transcript and protein product characterized. Although the functional properties of B. stearothermophilus and E. coli IF1, compared in several translational tests in the presence of both homologous and heterologous components, are not entirely identical, the two proteins are interchangeable in an in vitro translational system programmed with a natural mRNA. The availability of purified B. stearothermophilus IF1 now allows us to analyze the translation initiation pathway using efficient in vitro tests based entirely on purified components derived from this thermophilic Gram-positive bacterium.


Asunto(s)
Proteínas Bacterianas/genética , Factor 1 Eucariótico de Iniciación/genética , Geobacillus stearothermophilus/genética , Factor 1 Procariótico de Iniciación/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Northern Blotting , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Factor 1 Eucariótico de Iniciación/metabolismo , Geobacillus stearothermophilus/metabolismo , Datos de Secuencia Molecular , Iniciación de la Cadena Peptídica Traduccional , Factor 1 Procariótico de Iniciación/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido
15.
Mol Microbiol ; 70(6): 1368-77, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18976282

RESUMEN

Bacterial translation initiation factor IF1 is homologous to archaeal aIF1A and eukaryal eIF1A, which form a complex with their homologous IF2-like factors (aIF5B and eIF5B respectively) during initiation of protein synthesis. A similar IF1-IF2 interaction is assumed to occur in all bacteria and supported by cross-linking data and stabilization of the 30S-IF2 interaction by IF1. Here we compare Escherichia coli IF1 with thermophilic factors from Bacillus stearothermophilus and Thermus thermophilus. All three IF1s are structurally similar and functionally interchangeable in vivo and in vitro. However, the thermophilic factors do not stimulate ribosomal binding of IF2DeltaN, regardless of 30S subunits and IF2 origin. We conclude that an IF1-IF2 interaction is not universally conserved and is not essential for cell survival.


Asunto(s)
Escherichia coli/metabolismo , Geobacillus stearothermophilus/metabolismo , Factor 1 Procariótico de Iniciación/metabolismo , Factor 2 Procariótico de Iniciación/metabolismo , Thermus thermophilus/metabolismo , Escherichia coli/genética , Prueba de Complementación Genética , Geobacillus stearothermophilus/genética , Factor 1 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/genética , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Thermus thermophilus/genética
16.
Expert Opin Ther Targets ; 12(5): 519-34, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18410237

RESUMEN

BACKGROUND: Translation initiation is a basic and universal biological process that employs significantly different components and displays substantially different mechanisms in bacterial, archaeal and eukaryotic cells. A large amount of detailed mechanistic and structural information on the bacterial translation initiation apparatus has been uncovered in recent years. OBJECTIVE: to understand which translation initiation steps could represent a novel or underexploited target for the discovery of new and specific antibacterial drugs. METHODS: Brief descriptions of the properties and mechanism of action of the major antibiotics that have a documented direct inhibitory effect on bacterial translation initiation are presented. RESULTS/CONCLUSIONS: Considerations and predictions concerning a future scenario for research and identification of bacterial translation initiation inhibitors are presented.


Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Iniciación de la Cadena Peptídica Traduccional/fisiología , Factores de Iniciación de Péptidos/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo
17.
Methods Enzymol ; 430: 45-58, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17913634

RESUMEN

Three protocols to perform time-resolved in situ probing of rRNA are described. The three methods (chemical modification with DMS and rRNA backbone cleavage by hydroxyl radicals generated by either K-peroxonitrite or Fe(II)-EDTA) make use of a quench-flow apparatus and exploit reactions that are faster than the interactions of ribosomal subunits with their ligands. These methods allow the investigation of the path and dynamics, in a approximately equal 50 to 1500ms time range, of the binding and dissociation of ribosomal ligands.


Asunto(s)
Ligandos , Sondas Moleculares , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Alquilantes/química , Alquilantes/metabolismo , Ácido Edético/química , Ácido Edético/metabolismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Reproducibilidad de los Resultados , Ésteres del Ácido Sulfúrico/química , Ésteres del Ácido Sulfúrico/metabolismo , Factores de Tiempo
18.
Methods Enzymol ; 431: 229-67, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17923238

RESUMEN

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores de la Síntesis del Ácido Nucleico/aislamiento & purificación , Biosíntesis de Proteínas/efectos de los fármacos , Animales , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/metabolismo , Sistema Libre de Células/metabolismo , Células Cultivadas , Técnicas de Laboratorio Clínico , Humanos , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/análisis , Factor 2 Procariótico de Iniciación/antagonistas & inhibidores , Factor 2 Procariótico de Iniciación/fisiología , Proteínas de Unión a Caperuzas de ARN/fisiología , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Aminoacilación de ARN de Transferencia/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismo
19.
Nucleic Acids Res ; 35(18): 6330-7, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17881364

RESUMEN

The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. In accordance with previously published findings, we show that these motifs occur in AT-rich regions of DNA. On the basis of these observations, we propose that H-NS silences extensive regions of the bacterial chromosome by binding first to nucleating high-affinity sites and then spreading along AT-rich DNA. This spreading would be reinforced by the frequent occurrence of the motif in such regions. Our findings suggest that such an organization enables the silencing of extensive regions of the genetic material, thereby providing a coherent framework that unifies studies on the H-NS protein and a concrete molecular basis for the genetic control of H-NS transcriptional silencing.


Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , Proteínas de Unión al ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Genoma Bacteriano , Secuencia Rica en At , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Huella de ADN , ADN Bacteriano/metabolismo , Escherichia coli/genética , Redes Reguladoras de Genes , Islas Genómicas , Genómica , Operón , Proteobacteria/genética
20.
RNA ; 13(8): 1355-65, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17592046

RESUMEN

Expression of Escherichia coli infC, which encodes translation initiation factor IF3 and belongs to a transcriptional unit containing several promoters and terminators, is enhanced after cold shock, causing a transient increase of the IF3/ribosomes ratio. Here we show that after cold shock the two less used promoters (P(T) and P(I1)) remain active and/or are activated, resulting in de novo infC transcription and IF3 synthesis. These two events are partly responsible for the stoichiometric imbalance of the IF3/ribosomes ratio that contributes to establishing the cold-shock translational bias whereby cold-shock mRNAs are preferentially translated by cold-stressed cells while bulk mRNAs are discriminated against. Analysis of the IF3 functions at low temperature sheds light on the molecular mechanism by which IF3 contributes to the cold-shock translational bias. IF3 was found to cause a strong rate increase of fMet-tRNA binding to ribosomes programmed with cold-shock mRNA, an activity essential for the rapid formation of "30S initiation complexes" at low temperature. The increased IF3/ribosome ratio occurring during cold adaptation was also essential to overcome the higher stability of 70S monomers at low temperature so as to provide a sufficient pool of dissociated 30S subunits capable of "70S initiation complex" formation. Finally, at low temperature IF3 was shown to be endowed with the capacity of discriminating against translation of non-cold-shock mRNAs by a cold-shock-specific "fidelity" function operating with a mechanism different from those previously described, insofar as IF3 does not interfere with formation of 30S initiation complex containing these mRNAs, but induces the formation of nonproductive 70S initiation complexes.


Asunto(s)
Frío , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factor 3 Procariótico de Iniciación/genética , Escherichia coli/fisiología , ARN Mensajero , Ribosomas/fisiología
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