RESUMEN
Initiation factor (IF) 2 controls the fidelity of translation initiation by selectively increasing the rate of 50S ribosomal subunit joining to 30S initiation complexes (ICs) that carry an N-formyl-methionyl-tRNA (fMet-tRNAfMet). Previous studies suggest that rapid 50S subunit joining involves a GTP- and fMet-tRNAfMet-dependent "activation" of IF2, but a lack of data on the structure and conformational dynamics of 30S IC-bound IF2 has precluded a mechanistic understanding of this process. Here, using an IF2-tRNA single-molecule fluorescence resonance energy transfer signal, we directly observe the conformational switch that is associated with IF2 activation within 30S ICs that lack IF3. Based on these results, we propose a model of IF2 activation that reveals how GTP, fMet-tRNAfMet, and specific structural elements of IF2 drive and regulate this conformational switch. Notably, we find that domain III of IF2 plays a pivotal, allosteric, role in IF2 activation, suggesting that this domain can be targeted for the development of novel antibiotics.
Asunto(s)
Escherichia coli/fisiología , Factor 2 Procariótico de Iniciación/fisiología , Biosíntesis de Proteínas/fisiología , Subunidades Ribosómicas Grandes Bacterianas/fisiología , Subunidades Ribosómicas Pequeñas Bacterianas/fisiología , Regulación Alostérica/fisiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiología , Transferencia Resonante de Energía de Fluorescencia/métodos , Guanosina Trifosfato/metabolismo , Cinética , Modelos Biológicos , Mutación , Factor 2 Procariótico de Iniciación/química , Conformación Proteica , Dominios Proteicos/fisiología , ARN de Transferencia de Metionina/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Decades of extensive biochemical and biophysical research have outlined the mechanism of translation. Rich structural studies have provided detailed snapshots of the translational machinery at all phases of the translation cycle. However, the relationship between structural dynamics, composition, and function remains unknown. The multistep nature of each stage of the translation cycle results in rapid desynchronization of individual ribosomes, thus hindering elucidation of the underlying mechanisms by conventional bulk biophysical and biochemical methods. Single-molecule approaches unsusceptible to these complications have led to the first glances at both compositional and conformational dynamics on the ribosome and their impact on translational control. These experiments provide the necessary link between static structure and mechanism, often providing new perspectives. Here we review recent advances in the field and their relationship to structural and biochemical data.
Asunto(s)
Modelos Genéticos , Biosíntesis de Proteínas/fisiología , Ribosomas/fisiología , Biología Molecular/métodos , Conformación Molecular , Extensión de la Cadena Peptídica de Translación , Factor 2 Procariótico de Iniciación/fisiología , ARN de Transferencia/fisiología , Ribosomas/química , Ribosomas/ultraestructuraRESUMEN
In this paper, we describe a new mutation, gicD1, that gives a cold-sensitive phenotype in bacterial cell growth. Complementation analysis showed gicD1 to be allelic to infB. We identify gicD1 to be a valine to isoleucine substitution in initiation factor-2 (IF2) of a residue that seems to be well conserved in eubacterial IF2 proteins. This mutation lies in a region distant from the G-domain to which all earlier reported cold-sensitive mutations cluster. We describe a novel phenotype of the mutant that is suppression of rpsL31-mediated streptomycin resistance in cold. We provide evidence that mutant IF2 specifically interacts with rpsL31 in cold, leading to a bacteriostatic effect on host cells.
Asunto(s)
Frío , Escherichia coli/fisiología , Mutación Missense , Factor 2 Procariótico de Iniciación/fisiología , Estrés Fisiológico , Sustitución de Aminoácidos/genética , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli , Prueba de Complementación Genética , Factor 2 Procariótico de Iniciación/genética , Mapeo de Interacción de Proteínas , Proteínas Ribosómicas/genética , Estreptomicina/farmacología , Supresión GenéticaRESUMEN
The Escherichia coli protein YjeQ is a circularly permuted GTPase that is broadly conserved in bacteria. An emerging body of evidence, including cofractionation and in vitro binding to the ribosome, altered polysome profiles after YjeQ depletion, and stimulation of GTPase activity by ribosomes, suggests that YjeQ is involved in ribosome function. The growth of strains lacking YjeQ in culture is severely compromised. Here, we probed the cellular function of YjeQ with genetic screens of ordered E. coli genomic libraries for suppressors and enhancers of the slow-growth phenotype of a delta yjeQ strain. Screening for suppressors using an ordered library of 374 clones overexpressing essential genes and genes associated with ribosome function revealed that two GTPases, Era and initiation factor 2, ameliorated the growth and polysome defects of the delta yjeQ strain. In addition, seven bona fide enhancers of slow growth were identified (delta tgt, delta ksgA, delta ssrA, delta rimM, delta rluD, delta trmE/mnmE, and delta trmU/mnmA) among 39 deletions (in genes associated with ribosome function) that we constructed in the delta yjeQ genetic background. Taken in context, our work is most consistent with the hypothesis that YjeQ has a role in late 30S subunit biogenesis.
Asunto(s)
Proteínas de Escherichia coli/genética , Escherichia coli/genética , GTP Fosfohidrolasas/genética , Ribosomas/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/fisiología , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Mutación , Polirribosomas/metabolismo , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/metabolismo , Factor 2 Procariótico de Iniciación/fisiología , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiologíaRESUMEN
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/metabolismoRESUMEN
During initiation of bacterial protein synthesis, messenger RNA and fMet-tRNAfMet bind to the 30S ribosomal subunit together with initiation factors IF1, IF2, and IF3. Docking of the 30S preinitiation complex to the 50S ribosomal subunit results in a peptidyl-transfer competent 70S ribosome. Initiation with an elongator tRNA may lead to frameshift and an aberrant N-terminal sequence in the nascent protein. We show how the occurrence of initiation errors is minimized by (1) recognition of the formyl group by the synergistic action of IF2 and IF1, (2) uniform destabilization of the binding of all tRNAs to the 30S subunit by IF3, and (3) an optimal distance between the Shine-Dalgarno sequence and the initiator codon. We suggest why IF1 is essential for E. coli, discuss the role of the G-C base pairs in the anticodon stem of some tRNAs, and clarify gene expression changes with varying IF3 concentration in the living cell.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Factores Procarióticos de Iniciación/fisiología , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/metabolismo , ARN de Transferencia de Fenilalanina/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas Bacterianas/genética , Unión Competitiva , Cinética , Modelos Biológicos , Factor 1 Procariótico de Iniciación/fisiología , Factor 2 Procariótico de Iniciación/fisiología , Factor 3 Procariótico de Iniciación/fisiología , Factores Procarióticos de Iniciación/clasificación , ARN de Transferencia de Metionina/genética , ARN de Transferencia de Fenilalanina/genéticaRESUMEN
Three consecutive G-C pairs in the anticodon stem are a key discriminatory feature of initiator tRNA and are required for its selection by IF3. Here, we have mutated two 16S rRNA nucleotides, G1338 and A1339, which provide the sole contact to the G-C pairs of tRNA(fMet) bound to the ribosomal P site. We have tested their effects on translational activities in vivo and have affinity-purified mutant 30S subunits for functional analysis in vitro. Our results are consistent with the formation of Type II and I minor interactions, respectively, between G1338 and A1339 and the anticodon stem of tRNA and suggest that these interactions play a role in tRNA(fMet) discrimination by IF3. Moreover, our findings indicate that discrimination also involves recognition of at least one additional feature of the tRNA(fMet) anticodon stem loop.
Asunto(s)
Adenina/metabolismo , Guanina/metabolismo , ARN Ribosómico 16S/metabolismo , ARN de Transferencia de Metionina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica/fisiología , Mutación , Factor 2 Procariótico de Iniciación/fisiología , Factor 3 Procariótico de Iniciación/fisiología , Biosíntesis de Proteínas/genética , ARN Ribosómico 16S/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA. ifm1 mutants, lacking the mitochondrial initiation factor 2 (mIF2), are unable to respire, indicative of defective mitochondrial protein synthesis, but their respiratory defect could be complemented by plasmid-borne copies of either the yeast IFM1 gene or a cDNA encoding bovine mIF2. Moreover, the bovine mIF2 sustained normal respiration in ifm1 fmt1 double mutants. Bovine mIF2 supported the same pattern of mitochondrial translation products as yeast mIF2, and the pattern did not change in cells lacking formylated Met-tRNAfMet. Mutant yeast lacking any mIF2 retained the ability to synthesize low levels of a subset of mitochondrially encoded proteins. The ifm1 null mutant was used to analyze the domain structure of yeast mIF2. Contrary to a previous report, the C terminus of yeast mIF2 is required for its function in vivo, whereas the N-terminal domain could be deleted. Our results indicate that formylation of initiator methionyl-tRNA is not required for mitochondrial protein synthesis. The ability of bovine mIF2 to support mitochondrial translation in the yeast fmt1 mutant suggests that this phenomenon may extend to mammalian mitochondria as well.