RESUMEN
Transfer RNAs from all organisms contain many modified nucleosides. Their vastly different chemical structures, their presence in different tRNAs, their occurrence in different locations in tRNA and their influence on different reactions in which tRNA participates suggest that each modified nucleoside may have its own specific function. However, since the frequency of frameshifting in several different mutants [mnmA, mnmE, tgt, truA (hisT), trmD, miaA, miaB and miaE] defective in tRNA modification was higher compared with the corresponding wild-type controls, these modifications have a common function: they all improve reading frame maintenance. Frameshifting occurs by peptidyl-tRNA slippage, which is influenced by the hypomodified tRNA in two ways: (i) a hypomodified tRNA in the ternary complex may decrease the rate by which the complex is recruited to the A-site and thereby increasing peptidyl-tRNA slippage; or (ii) a hypomodified peptidyl-tRNA may be more prone to slip than its fully modified counterpart. We propose that the improvement of reading frame maintenance has been and is the major selective factor for the emergence of new modified nucleosides.
Asunto(s)
Escherichia coli/genética , Mutación del Sistema de Lectura , Modelos Genéticos , ARN de Transferencia Aminoácido-Específico/genética , Aminoacil-ARN de Transferencia/genética , ARN de Transferencia/genética , Sistemas de Lectura , Salmonella typhimurium/genética , Secuencia de Bases , Codón/genética , Genotipo , Oligodesoxirribonucleótidos/química , Fenotipo , ARN Bacteriano/genética , ARN de Transferencia/química , Aminoacil-ARN de Transferencia/química , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Fenilalanina/genética , ARN de Transferencia de Prolina/genética , ARN de Transferencia de Valina/genética , Valores de Referencia , beta-Galactosidasa/genética , beta-Lactamasas/genéticaRESUMEN
The evolution of reading frame maintenance must have been an early event, and presumably preceded the emergence of the three domains Archaea, Bacteria and Eukarya. Features evolved early in reading frame maintenance may still exist in present-day organisms. We show that one such feature may be the modified nucleoside 1-methylguanosine (m(1)G37), which prevents frameshifting and is present adjacent to and 3' of the anticodon (position 37) in the same subset of tRNAs from all organisms, including that with the smallest sequenced genome (Mycoplasma genitalium), and organelles. We have identified the genes encoding the enzyme tRNA(m(1)G37)methyltransferase from all three domains. We also show that they are orthologues, and suggest that they originated from a primordial gene. Lack of m(1)G37 severely impairs the growth of a bacterium and a eukaryote to a similar degree. Yeast tRNA(m(1)G37)methyltransferase also synthesizes 1-methylinosine and participates in the formation of the Y-base (yW). Our results suggest that m(1)G37 existed in tRNA before the divergence of the three domains, and that a tRNA(m(1)G37)methyltrans ferase is part of the minimal set of gene products required for life.
Asunto(s)
Archaea/genética , Bacterias/genética , Evolución Biológica , Mycoplasma/genética , ARN Bacteriano/genética , ARN de Transferencia/genética , Sistemas de Lectura , Salmonella typhimurium/genética , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Genoma Bacteriano , Humanos , Datos de Secuencia Molecular , Salmonella typhimurium/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , ARNt Metiltransferasas/químicaRESUMEN
Induction of GCN4 translation in amino acid-starved cells involves the inhibition of initiator tRNA(Met) binding to eukaryotic translation initiation factor 2 (eIF2) in response to eIF2 phosphorylation by protein kinase GCN2. It was shown previously that GCN4 translation could be induced independently of GCN2 by overexpressing a mutant tRNA(AAC)(Val) (tRNA(Val*)) or the RNA component of RNase MRP encoded by NME1. Here we show that overexpression of the tRNA pseudouridine 55 synthase encoded by PUS4 also leads to translational derepression of GCN4 (Gcd(-) phenotype) independently of eIF2 phosphorylation. Surprisingly, the Gcd(-) phenotype of high-copy-number PUS4 (hcPUS4) did not require PUS4 enzymatic activity, and several lines of evidence indicate that PUS4 overexpression did not diminish functional initiator tRNA(Met) levels. The presence of hcPUS4 or hcNME1 led to the accumulation of certain tRNA precursors, and their Gcd(-) phenotypes were reversed by overexpressing the RNA component of RNase P (RPR1), responsible for 5'-end processing of all tRNAs. Consistently, overexpression of a mutant pre-tRNA(Tyr) that cannot be processed by RNase P had a Gcd(-) phenotype. Interestingly, the Gcd(-) phenotype of hcPUS4 also was reversed by overexpressing LOS1, required for efficient nuclear export of tRNA, and los1Delta cells have a Gcd(-) phenotype. Overproduced PUS4 appears to impede 5'-end processing or export of certain tRNAs in the nucleus in a manner remedied by increased expression of RNase P or LOS1, respectively. The mutant tRNA(Val*) showed nuclear accumulation in otherwise wild-type cells, suggesting a defect in export to the cytoplasm. We propose that yeast contains a nuclear surveillance system that perceives defects in processing or export of tRNA and evokes a reduction in translation initiation at the step of initiator tRNA(Met) binding to the ribosome.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas Fúngicas/genética , Proteínas Quinasas/genética , ARN de Transferencia de Metionina/genética , Proteínas de Saccharomyces cerevisiae , Oxidorreductasas de Alcohol , Aminohidrolasas , Secuencia de Bases , Transporte Biológico/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Hibridación Fluorescente in Situ , Transferasas Intramoleculares/genética , Datos de Secuencia Molecular , Mutación , Fosforilación , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Pirofosfatasas , Factores de Transcripción/genética , Levaduras/metabolismoRESUMEN
Full expression of the virulence genes of Shigella flexneri requires the presence of two modified nucleosides in the tRNA [queuosine, Q34, present in the wobble position (position 34) and 2-methylthio-N6-isopentenyladenosine (ms2i6A37, adjacent to and 3' of the anticodon)]. The synthesis of these two nucleosides depends on the products of the tgt and miaA genes respectively. We have shown that the intracellular concentration of the virulence-related transcriptional regulator VirF is reduced in the absence of either of these modified nucleosides. The intracellular concentration of VirF is correlated with the expression of the virulence genes. Overproduction of VirF in the tgt and the miaA mutants suppressed the less virulent (tgt) or the avirulent (miaA) phenotypes respectively, caused by the tRNA modification deficiency. This suggests that the primary result of undermodification of the tRNA is a poor translation of virF mRNA and not of any other mRNA whose product acts downstream of the action of VirF. Shigella showed no virulence phenotypes at 30 degrees C, but forced synthesis of VirF at 30 degrees C induced the virulence phenotype at this low temperature. In addition, removal of the known gene silencer H-NS by a mutation in its structural gene hns increased the synthesis of VirF at low temperature and thus induced a virulent phenotype at 30 degrees C. Conversely, decreased expression of VirF at 37 degrees C induced by the addition of novobiocin, a known inhibitor of gyrase, led to an avirulent phenotype. We conclude that tRNA modification, temperature and superhelicity have the same target - the expression of VirF - to influence the expression of the central regulatory gene virB and thereby the virulence of Shigella. These results further strengthen the suggestion that the concentration of VirF is the critical factor in the regulation of virulence in Shigella. In addition, they emphasize the role of the bacterial translational machinery in the regulation of the expression of virulence genes which appears here quantitatively as important as the well-established regulation on the transcriptional level.
Asunto(s)
Proteínas Bacterianas/genética , ADN Superhelicoidal/química , ARN de Transferencia/metabolismo , Shigella flexneri/genética , Factores de Virulencia , Antígenos Bacterianos/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN de Transferencia/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidad , Temperatura , Transcripción Genética , Virulencia/genéticaRESUMEN
The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleoside N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of miaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of the miaB gene, at which the majority (96%) of the miaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation.
Asunto(s)
Escherichia coli/genética , ARN de Transferencia/genética , Salmonella typhimurium/genética , Sulfurtransferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Cromatografía Líquida de Alta Presión , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Prueba de Complementación Genética , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/genética , Isopenteniladenosina/metabolismo , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis , Sistemas de Lectura Abierta , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Salmonella typhimurium/metabolismo , Homología de Secuencia de Aminoácido , Sulfurtransferasas/fisiología , Transcripción GenéticaRESUMEN
Transfer RNA modification improves the rate of aa-tRNA selection at the A-site and the fitness in the P-site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471-482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed.
Asunto(s)
Sistema de Lectura Ribosómico/fisiología , ARN de Transferencia/metabolismo , Sistema de Lectura Ribosómico/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN de Transferencia/genéticaRESUMEN
In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG. These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37. The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs. We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell. Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA. However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase. Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme. We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA. Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.
Asunto(s)
Salmonella typhimurium/enzimología , ARNt Metiltransferasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , División Celular/genética , Mutación del Sistema de Lectura/genética , Guanosina/análogos & derivados , Guanosina/análisis , Guanosina/genética , Metilación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fenotipo , ARN de Transferencia de Prolina/genética , ARN de Transferencia de Prolina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , ARNt Metiltransferasas/químicaRESUMEN
Most missense errors have little effect on protein function, since they only exchange one amino acid for another. However, processivity errors, frameshifting or premature termination result in a synthesis of an incomplete peptide. There may be a connection between missense and processivity errors, since processivity errors now appear to result from a second error occurring after recruitment of an errant aminoacyl-tRNA, either spontaneous dissociation causing premature termination or translational frameshifting. This is clearest in programmed translational frameshifting where the mRNA programs errant reading by a near-cognate tRNA; this error promotes a second frameshifting error (a dual-error model of frameshifting). The same mechanism can explain frameshifting by suppressor tRNAs, even those with expanded anticodon loops. The previous model that suppressor tRNAs induce quadruplet translocation now appears incorrect for most, and perhaps for all of them. We suggest that the 'spontaneous' tRNA-induced frameshifting and 'programmed' mRNA-induced frameshifting use the same mechanism, although the frequency of frameshifting is very different. This new model of frameshifting suggests that the tRNA is not acting as the yardstick to measure out the length of the translocation step. Rather, the translocation of 3 nucleotides may be an inherent feature of the ribosome.
Asunto(s)
Biosíntesis de Proteínas , Sistemas de Lectura , Emparejamiento Base , Secuencia de Bases , Mutación del Sistema de Lectura , Modelos Genéticos , Mutación Missense , Conformación de Ácido Nucleico , ARN de Transferencia/genética , Ribosomas/metabolismoRESUMEN
Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression of GCN4 mRNA. The phenotypes of gcd10 mutants were suppressed by high-copy-number IMT genes, encoding initiator methionyl tRNA (tRNAiMet), or LHP1, encoding the yeast homolog of the human La autoantigen. The gcd10-504 mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of a GCD10 deletion was suppressed by high-copy-number IMT4, indicating that its role in expression of mature tRNAiMet is the essential function of Gcd10p. A gcd14-2 mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMet processing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMet maturation. The chromatographic behavior of elongator and initiator tRNAMet on a RPC-5 column indicated that both species are altered structurally in gcd10Delta cells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable in gcd10Delta tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.
Asunto(s)
Adenosina/análogos & derivados , Proteínas Nucleares/metabolismo , Iniciación de la Cadena Peptídica Traduccional , Procesamiento Postranscripcional del ARN , ARN de Transferencia de Metionina/metabolismo , Adenosina/biosíntesis , Genes Fúngicos , Mutación , Proteínas Nucleares/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Supresión GenéticaRESUMEN
According to the prevailing model, frameshift-suppressing tRNAs with an extra nucleotide in the anticodon loop suppress +1 frameshift mutations by recognizing a four-base codon and promoting quadruplet translocation. We present three sets of experiments that suggest a general alternative to this model. First, base modification should actually block such a four-base interaction by two classical frameshift suppressors. Second, for one Salmonella suppressor tRNA, it is not mutant tRNA but a structurally normal near cognate that causes the +1 shift in-frame. Finally, frameshifting occurs in competition with normal decoding of the next in-frame codon, consistent with an event that occurs in the ribosomal P site after the translocation step. These results suggest an alternative model involving peptidyl-tRNA slippage at the classical CCC-N and GGG-N frameshift suppression sites.
Asunto(s)
Anticodón/química , Mutación del Sistema de Lectura/genética , Saccharomyces cerevisiae/genética , Salmonella typhimurium/genética , Anticodón/genética , Cartilla de ADN , Regulación Bacteriana de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Guanosina/análogos & derivados , Guanosina/genética , Conformación de Ácido Nucleico , Fenotipo , Biosíntesis de Proteínas/genética , ARN Mensajero/química , ARN de Transferencia/químicaRESUMEN
The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(i.v. Ser) in Escherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E. coli. Mutants (miaE) of S. typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.
Asunto(s)
Ciclo del Ácido Cítrico , Isopenteniladenosina/análogos & derivados , ARN de Transferencia/química , ARN de Transferencia/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Mapeo Cromosómico , Fumaratos/metabolismo , Genes Bacterianos , Prueba de Complementación Genética , Isopenteniladenosina/química , Malatos/metabolismo , Mutación , Fenotipo , ARN de Transferencia/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Ácido Succínico/metabolismoRESUMEN
tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.
Asunto(s)
Adenosina/análogos & derivados , Escherichia coli/genética , ARN Bacteriano/química , ARN de Transferencia de Treonina/química , Adenosina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Codón , Escherichia coli/crecimiento & desarrollo , Datos de Secuencia Molecular , Operón , ARN Bacteriano/metabolismo , ARN de Transferencia de Treonina/metabolismo , Relación Estructura-Actividad , ARNt Metiltransferasas/genéticaRESUMEN
The virulence of the human pathogen Shigella flexneri is dependent on both chromosome- and large-virulence-plasmid-encoded genes. A kanamycin resistance cassette mutation in the miaA gene (miaA::Km Sma), which encodes the tRNA N6-isopentyladenosine (i6A37) synthetase and is involved in the first step of the synthesis of the modified nucleoside 2-methylthio-N6-isopentenyladenosine (ms2i6A), was transferred to the chromosome of S. flexneri 2a by phage P1 transduction. In the wild-type bacterium, ms2i6A37 is present in position 37 (next to and 3' of the anticodon) in a subset of tRNA species-reading codons starting with U (except tRNA(Ser) species SerI and SerV). The miaA::Km Sma mutant of S. flexneri accordingly lacked ms2i6A37 in its tRNA. In addition, the mutant strains showed reduced expression of the virulence-related genes ipaB, ipaC, ipaD, virG, and virF, accounting for sixfold-reduced contact hemolytic activity and a delayed response in the focus plaque assay. A cloned sequence resulting from PCR amplification of the wild-type Shigella chromosome and exhibiting 99% homology with the nucleotide sequence of the Escherichia coli miaA gene complemented the virulence-associated phenotypes as well as the level of the modified nucleoside ms2i6A in the tRNA of the miaA mutants. In the miaA mutant, the level of the virulence-associated protein VirF was reduced 10-fold compared with the wild type. However, the levels of virF mRNA were identical in the mutant and in the wild type. These findings suggest that a posttranscriptional mechanism influenced by the presence of the modified nucleoside ms2i6A in the tRNA is involved in the expression of the virF gene. The role of the miaA gene in the virulence of other Shigella species and in enteroinvasive E. coli was further generalized.
Asunto(s)
Transferasas Alquil y Aril , Regulación Bacteriana de la Expresión Génica , Isopenteniladenosina/análogos & derivados , ARN de Transferencia/fisiología , Shigella flexneri/genética , Transferasas/fisiología , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas de Unión al ADN/genética , Isopenteniladenosina/metabolismo , Datos de Secuencia Molecular , ARN Bacteriano/fisiología , Shigella flexneri/patogenicidad , Factores de Transcripción/genéticaRESUMEN
In Salmonella typhimurium seven tRNA species specific for leucine, proline and arginine have 1-methylguanosine (m1G) next to and 3' of the anticodon (position 37 of tRNA), five tRNA species specific for phenylalanine, serine, tyrosine, cysteine and tryptophan have 2-methylthio-N-6-(cis-hydroxy)isopentenyladenosine (ms2io6A) in the same position of the tRNA, and four tRNA species, specific for leucine and proline, have pseudouridine (Psi) as the last 3' nucleotide in the anticodon loop (position 38) or in the anticodon stem (positions 39 and 40). Mutants deficient in the synthesis of these modified nucleosides have been used to study their role in the first step of translation elongation, i.e. the aa-tRNA selection step in which the ternary complex (EF-Tu-GTP-aa-tRNA) binds at the cognate codon in the A-site on the mRNA programmed ribosome. We have found that the Psi present in the anticodon loop (position 38) stimulates the selection of tRNA specific for leucine whereas Psi in the anticodon stem did not affect the selection of tRNA specific for proline. The m1G37 strongly stimulates the rate of selection of the three tRNA species specific for proline and one tRNA species specific for arginine but has only minor or no effect on the selection of the three tRNA species specific for leucine. Likewise, the ms2io6A, present in the same position as m1G37 but in another subset of tRNA species, stimulates the selection of tRNA specific for tyrosine, stimulates to some extent also tRNA species specific for cysteine and tryptophan, but has no influence on the rate of selection of tRNA specific for phenylalanine. We conclude that function of m1G and ms2io6A present next to and 3' of the anticodon influences the in vivo aa-tRNA selection in a tRNA-dependent manner.
Asunto(s)
Anticodón , Guanosina/análogos & derivados , Conformación de Ácido Nucleico , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Salmonella typhimurium/metabolismo , Secuencia de Bases , Sitios de Unión , Codón , Mutación del Sistema de Lectura , Genotipo , Guanosina/análisis , Guanosina Trifosfato/metabolismo , Modelos Estructurales , Factor Tu de Elongación Peptídica/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Arginina/química , ARN de Transferencia de Arginina/metabolismo , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Prolina/química , ARN de Transferencia de Prolina/metabolismo , Ribosomas/metabolismo , Salmonella typhimurium/genética , beta-Galactosidasa/biosíntesisRESUMEN
Maturation of tRNA and rRNA and the assembly of the ribosome in all organisms occurs in vivo in a complex pathway in which various proteins such as endo- and exonucleases, tRNA and rRNA modifying enzymes and ribosomal proteins, act concomitantly and temporarily during the maturation process. One class of RNA binding proteins are the tRNA modifying enzymes, which catalyse the formation of various modified nucleosides present in tRNA. Here we analyse the consequences of various alterations in a tRNA on the formation of modified nucleosides in the tRNA and the aminoacylation of it under true in vivo conditions, i.e. in a cell with normal amounts of the tRNA substrate and the tRNA binding protein. We have devised a selection method to obtain mutants of tRNA(Pro)GGG in Salmonella typhimurium that may no longer be a substrate inl vivo for the tRNA(m1G37)methyltransferase. These mutant tRNAs were purified from cells in balanced growth by a solid phase hybridisation technique and the presence of 1-methylguanosine (m1G) in position 37 next to the anticodon was monitored. Of 13 different mutant tRNA(Pro)GGG species analysed, eight of them had a drastically reduced level of m1G. Some of these mutant tRNA species had alterations far from the nucleotide G37 modified by the enzyme; e.g. base-pair disruptions in the first, fourth and eighth (last) base-pair of the acceptor stem, in the D-stem, and in the top of the anticodon stem. The structure of all the mutant tRNA(Pro)GGG species must deviate from the wild-type form, since they all induced +1 frameshifting. Still, tRNA(Pro)GGG from five of the mutants had normal levels of m1G. Thus, only a subset of mutations, all inducing an altered tRNA structure, resulted in m1G deficiency. However, those alterations in tRNA(Pro)GGG, which influenced the tRNA(m1G37)methyltransferase activity, did not affect in vivo the formation of four other modified nucleosides and the aminoacylation of tRNA(Pro)GGG, demonstrating the extreme dependence of the tRNA(m1G37)methyltransferase on an almost perfect three-dimensional structure of the tRNA. We discuss that the conformation of the anticodon loop may be a major determining element for the formation of m1G37 in vivo.
Asunto(s)
Guanosina/análogos & derivados , ARN Bacteriano/metabolismo , ARN de Transferencia de Prolina/química , ARN de Transferencia de Prolina/genética , Salmonella typhimurium/genética , Secuencia de Bases , ADN Bacteriano/genética , Mutación del Sistema de Lectura , Guanosina/genética , Hibridación in Situ/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Precursores del ARN/genética , ARN Bacteriano/química , ARN Bacteriano/genética , ARN de Transferencia de Prolina/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Supresión Genética , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
A total of 12 Salmonella typhimurium mutants were selected with mutations in the minor tRNAProGGG which suppress a +1 frameshift mutation in the hisD gene. This tRNA normally has 1-methylguanosine (m1G37) next to and 3' of the anticodon (position 37). Since the presence of m1G37 prevents frameshifting, some of the +1 frameshift suppressor derivatives of tRNAProGGG had alterations in the primary sequence abolishing the formation of m1G37. However, several of the mutant tRNAProGGG species had a normal level of m1G37 and a normal-sized anticodon loop, showing that neither m1G37 deficiency, nor an oversized anticodon loop, is a prerequisite for +1 frameshifting. Moreover, base substitutions far from the anticodon, e.g. in the acceptor stem, DHU-loop and stem, and at the top of the anticodon stem, promoted +1 frameshifting. When the frameshifting site (CCC-Uaa; CCC is in the zero frame and a +1 frameshift moves the ribosome to the CC-U codon) is overlapped by a nonsense codon (UAA), the efficiency of frameshifting decreased when release factor 1 was over-expressed and increased at an elevated temperature in a mutant with a temperature-sensitive release factor 1. The frameshifting site (CCC-Uac) was also overlapped with the sense codon UAC, which is decoded by a tRNA species having a 2-methylthio-cis ribozeatin (ms2io6A) at position 37. Mutations in the miaA gene affect the formation of this modified nucleoside and result in an A instead of ms2io6A37 in the tRNA. Such an undermodified tRNA is very inefficient in translation and the efficiency of frameshifting increased in a miaA1 mutant. These results suggest that the frameshifting event occurs at the P-site, since the efficiency of frameshifting was sensitive to the decoding activity of the overlapping codon. We conclude that tRNA with mutations far from the anticodon, with a normal-sized anticodon loop and having m1G37 induce +1 frameshifting at the P-site.
Asunto(s)
Oxidorreductasas de Alcohol , Anticodón , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Mutación del Sistema de Lectura , Extensión de la Cadena Peptídica de Translación , ARN Bacteriano/genética , Aminoacil-ARN de Transferencia/genética , ARN de Transferencia de Prolina/genética , Salmonella typhimurium/genética , Secuencia de Bases , Cromosomas Bacterianos/genética , ADN Bacteriano/química , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Código Genético , Guanosina/análogos & derivados , Guanosina/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Conformación de Ácido Nucleico , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , TemperaturaRESUMEN
A temperature-sensitive mutant of Salmonella typhimurium LT2 was isolated. At the nonpermissive temperature cell division stopped and multinucleated filaments were formed. DNA, RNA, or protein synthesis was not affected until after about two generations. Different physiological conditions, such as anaerobiosis and different growth media, suppress the division deficiency at high temperatures. Certain mutations causing a reduced polypeptide chain elongation rate also suppress the division deficiency. The mutation is recessive and shown to be in the structural gene for release factor I (prfA). DNA sequencing of both the wild-type (prfA+) and mutant (prfA101) allele revealed a GC-to-AT transition in codon 168. Like other known prfA mutants, prfA101 can suppress amber mutations. The division defect in the prfA101 mutant strain could not be suppressed by overexpression of the ftsQAZ operon. Moreover, at the nonpermissive temperature the mutant shows a normal heat shock and SOS response and has a normal ppGpp level. We conclude that the prfA101-mediated defect in cell division is not directed through any of these metabolic pathways, which are all known to affect cell division. We speculate that the altered release factor I induces aberrant synthesis of an unidentified protein(s) involved in the elaborate process of septation.
Asunto(s)
Genes Bacterianos , Factores de Terminación de Péptidos/fisiología , Salmonella typhimurium/crecimiento & desarrollo , Aldehído Oxidorreductasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , División Celular/genética , División Celular/fisiología , Mapeo Cromosómico , Cromosomas Bacterianos , Genes Recesivos , Guanosina Tetrafosfato/metabolismo , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Mutación , Factores de Terminación de Péptidos/genética , Fenotipo , Biosíntesis de Proteínas , Respuesta SOS en Genética , Salmonella typhimurium/genética , Temperatura , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg). A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase. The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C. The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA. Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu). Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent. The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs. 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT. Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected. We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog. Our results also suggest that tRNA modification may be a regulatory device for gene expression.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Codón/genética , Guanosina/análogos & derivados , Aminoacil-ARN de Transferencia/química , Salmonella typhimurium/metabolismo , 3-Isopropilmalato Deshidrogenasa , Oxidorreductasas de Alcohol/genética , Aminoácidos/farmacología , Proteínas Bacterianas/genética , Carbono/metabolismo , Farmacorresistencia Microbiana , Inducción Enzimática , Regulación Bacteriana de la Expresión Génica/genética , Guanosina/fisiología , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Mutación , Operón/genética , Extensión de la Cadena Peptídica de Translación/genética , ARN Mensajero/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Supresión Genética , Tirosina/análogos & derivados , Tirosina/farmacología , beta-Galactosidasa/biosíntesis , ARNt Metiltransferasas/genéticaRESUMEN
The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , ARN/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Endorribonucleasas/biosíntesis , Escherichia coli/genética , Genes Bacterianos , Genotipo , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Plásmidos , ARN/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated. The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine. Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA. Only small amounts of io6A37 was found. We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37. The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes. The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain. The polypeptide chain elongation rate was also unaffected in the mutant. However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons. The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA. However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.