RESUMO
Translation of the isoleucine codon AUA in most prokaryotes requires a modified C (lysidine or agmatidine) at the wobble position of tRNA2(Ile) to base pair specifically with the A of the AUA codon but not with the G of AUG. Recently, a Bacillus subtilis strain was isolated in which the essential gene encoding tRNA(Ile)-lysidine synthetase was deleted for the first time. In such a strain, C34 at the wobble position of tRNA2(Ile) is expected to remain unmodified and cells depend on a mutant suppressor tRNA derived from tRNA1(Ile), in which G34 has been changed to U34. An important question, therefore, is how U34 base pairs with A without also base pairing with G. Here, we show (i) that unlike U34 at the wobble position of all B. subtilis tRNAs of known sequence, U34 in the mutant tRNA is not modified, and (ii) that the mutant tRNA binds strongly to the AUA codon on B. subtilis ribosomes but only weakly to AUG. These in vitro data explain why the suppressor strain displays only a low level of misreading AUG codons in vivo and, as shown here, grows at a rate comparable to that of the wild-type strain.
Assuntos
Bacillus subtilis/genética , Códon , Isoleucina/metabolismo , Biossíntese de Proteínas , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/metabolismo , Aminoacil-tRNA Sintetases/genética , Bacillus subtilis/crescimento & desenvolvimento , Deleção de Genes , Fenótipo , RNA de Transferência de Isoleucina/isolamento & purificação , Ribossomos/metabolismo , Aminoacilação de RNA de TransferênciaRESUMO
Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.
Assuntos
Anticódon/química , Códon/química , Isoleucina-tRNA Ligase/química , Mycoplasma/genética , RNA de Transferência de Isoleucina/química , Sequência de Aminoácidos , Arginina/química , Isoleucina-tRNA Ligase/metabolismo , Cinética , Dados de Sequência Molecular , Mycoplasma/enzimologia , RNA de Transferência de Isoleucina/isolamento & purificação , RNA de Transferência de Isoleucina/metabolismo , Ribossomos/metabolismo , Alinhamento de SequênciaRESUMO
A combination of hydrophobic chromatography on phenyl-Sepharose and reversed phase HPLC was used to purify individual tRNAs with high specific activity. The efficiency of chromatographic separation was enhanced by biochemical manipulations of the tRNA molecule, such as aminoacylation, formylation of the aminoacyl moiety and enzymatic deacylation. Optimal combinations are presented for three different cases. (i) tRNA(Phe) from Escherichia coli. This species was isolated by a combination of low pressure phenyl-Sepharose hydrophobic chromatography with RP-HPLC. (ii) tRNA(Ile) from E. coli: Aminoacylation increases the retention time for this tRNA in RP-HPLC. The recovered acylated intermediate is deacylated by reversion of the aminoacylation reaction and submitted to a second RP-HPLC run, in which deacylated tRNA(Ile) is recovered with high specific activity. (iii) tRNA(i)(Met) from Saccharomyces cerevisiae. The aminoacylated form of this tRNA is unstable. To increase stability, the aminoacylated form was formylated using E.coli: enzymes and, after one RP-HPLC step, the formylated derivative was deacylated using peptidyl-tRNA hydrolase from E.COLI: The tRNA(i)(Met) recovered after a second RP-HPLC run exhibited electrophoretic homogeneity and high specific activity upon aminoacylation. These combinations of chromatographic separation and biochemical modification can be readily adapted to the large-scale isolation of any particular tRNA.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia/métodos , RNA de Transferência/isolamento & purificação , Acilação , Hidrolases de Éster Carboxílico/isolamento & purificação , Hidrolases de Éster Carboxílico/metabolismo , Cromatografia em Agarose , Escherichia coli/genética , RNA Bacteriano/isolamento & purificação , RNA Fúngico/isolamento & purificação , RNA de Transferência/química , RNA de Transferência de Isoleucina/isolamento & purificação , RNA de Transferência de Metionina/isolamento & purificação , RNA de Transferência de Fenilalanina/isolamento & purificação , Saccharomyces cerevisiae/genética , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Bacillus subtilis, which belongs to Gram-positive eubacteria, has been predicted to have a minor isoleucine tRNA transcribed from the gene possessing the CAT anticodon, which corresponds to methionine. We isolated this tRNA and determined its sequence including modified nucleotides. Modified nucleotide analyses using TLC, UV, and FAB mass spectroscopy revealed that the first letter of the anticodon is modified to lysidine [4-amino-2-(N6-lysino)-1-beta-d-ribofuranosyl pyrimidine]. As a result, this tRNA agrees with the minor one predicted from the DNA sequence and is thought to decode the isoleucine codon AUA.
Assuntos
Anticódon/química , Bacillus subtilis/genética , DNA Bacteriano/genética , Metionina/genética , RNA de Transferência de Isoleucina/genética , Acilação , Anticódon/genética , Sequência de Bases , DNA Bacteriano/química , Genes Bacterianos/genética , Isoleucina/metabolismo , Lisina/análogos & derivados , Lisina/análise , Metionina/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleosídeos de Pirimidina/análise , RNA de Transferência de Isoleucina/isolamento & purificação , RNA de Transferência de Isoleucina/metabolismo , Análise de Sequência de RNA , Transcrição GênicaRESUMO
Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).
Assuntos
Bombyx/metabolismo , RNA de Transferência de Isoleucina/química , Fatores de Transcrição TFII , Fatores de Transcrição/química , Animais , Sequência de Bases , Bombyx/genética , Núcleo Celular/metabolismo , Cromatografia em Camada Fina , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos , Regiões Promotoras Genéticas , RNA Polimerase III/metabolismo , RNA de Transferência de Isoleucina/biossíntese , RNA de Transferência de Isoleucina/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaAssuntos
Cloroplastos/análise , Euglena gracilis/genética , Genes , RNA de Transferência/genética , Proteínas Ribossômicas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , RNA de Transferência/isolamento & purificação , RNA de Transferência de Cisteína/genética , RNA de Transferência de Cisteína/isolamento & purificação , RNA de Transferência de Isoleucina/genética , RNA de Transferência de Isoleucina/isolamento & purificação , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificaçãoRESUMO
A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium ("lysidine"), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.
