RESUMO
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.
Assuntos
Escherichia coli/genética , RNA de Transferência/genética , Salmonella typhimurium/genética , Sulfurtransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Cromatografia Líquida de Alta Pressão , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Genes Bacterianos , Teste de Complementação Genética , Isopenteniladenosina/análogos & derivados , Isopenteniladenosina/genética , Isopenteniladenosina/metabolismo , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese , Fases de Leitura Aberta , Plasmídeos/genética , Reação em Cadeia da Polimerase , Salmonella typhimurium/metabolismo , Homologia de Sequência de Aminoácidos , Sulfurtransferases/fisiologia , Transcrição GênicaRESUMO
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.
Assuntos
Anticódon , Guanosina/análogos & derivados , Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismo , Salmonella typhimurium/metabolismo , Sequência de Bases , Sítios de Ligação , Códon , Mutação da Fase de Leitura , Genótipo , Guanosina/análise , Guanosina Trifosfato/metabolismo , Modelos Estruturais , Fator Tu de Elongação de Peptídeos/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência de Arginina/química , RNA de Transferência de Arginina/metabolismo , RNA de Transferência de Leucina/química , RNA de Transferência de Leucina/metabolismo , RNA de Transferência de Prolina/química , RNA de Transferência de Prolina/metabolismo , Ribossomos/metabolismo , Salmonella typhimurium/genética , beta-Galactosidase/biossínteseRESUMO
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.
Assuntos
Anticódon/química , Anticódon/genética , Isopenteniladenosina/análogos & derivados , RNA Bacteriano/genética , RNA de Transferência Aminoácido-Específico/química , RNA de Transferência Aminoácido-Específico/genética , Sequência de Bases , Códon/genética , Genes Bacterianos , Isopenteniladenosina/química , Dados de Sequência Molecular , Estrutura Molecular , Mutação , RNA Bacteriano/química , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Supressão GenéticaRESUMO
Alagille syndrome (AS) is the second most frequent cause of intrahepatic cholestasis in children under one year of age. The disease has an autosomal dominant mode of inheritance with reduced penetrance and variable expressivity. It has been stated that this form of biliary paucity has a good long-term prognosis, but recent studies have revealed that the syndrome may be accompanied by long-term manifestations extending beyond childhood. Based on a case story the syndrome is described, and the diagnostic criteria, treatment and prognosis of AS are discussed.
Assuntos
Síndrome de Alagille/diagnóstico , Síndrome de Alagille/genética , Humanos , Lactente , Recém-Nascido , Masculino , PrognósticoRESUMO
Isopentenyl adenosine derivatives can be found next to the anticodon (position 37) in tRNA from both the Bacteria and Eucarya domains. These modified nucleosides improve the efficiency of tRNA in translation, can increase and decrease translational fidelity, and make the tRNA less codon context sensitive. In bacteria the synthesis of isopentenyl adenosine derivatives seems to be linked to iron metabolism and central metabolic pathways.