RESUMO
It is generally accepted that posttranscriptional modifications in RNA play a role in the fine-tuning of RNA function and the maintenance of RNA structure. This article describes the characterization of the posttranscriptional modifications in Legionella pneumophila 16S rRNA by mass spectrometry and reverse transcriptase assays. Eight modified nucleotides were identified and mapped in the 16S rRNA sequence. Situation of these data in relation to general 16S rRNA modification patterns shows that L. pneumophila is relatively less modified, and that the majority of the L. pneumophila 16S rRNA modifications are conserved among the bacteria characterized so far (Escherichia coli, Clostridium acetobutylicum, Thermus thermophilus, and Thermotoga maritima).
Assuntos
Legionella pneumophila/genética , RNA Ribossômico 16S/química , Cromatografia Líquida , Espectrometria de Massas , Conformação Molecular , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/isolamento & purificação , RNA Ribossômico 16S/metabolismoRESUMO
Post-transcriptional modifications in ribosomal RNA are believed to fine-tune the RNA functions. The present study describes the characterization of the post-transcriptional modifications in Clostridium acetobutylicum 16S rRNA, using high-pressure liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry and reverse transcriptase assays. The combination of these techniques allowed the identification of eleven modified nucleosides, which were mapped onto the rRNA sequence. The C. acetobutylicum modification map is similar to that of Escherichia coli, with the majority of the modifications near functionally important sites in the rRNA. Although, in general, the number of modifications in rRNA is smaller than in tRNA, the conservation of the modification sites seems to indicate that the post-transcriptional modifications in 16S rRNA provide a necessary prerequisite for the ribosomal function.
Assuntos
Clostridium acetobutylicum/genética , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Clostridium acetobutylicum/metabolismo , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleosídeos/química , Pseudouridina/química , RNA Ribossômico 16S/metabolismo , DNA Polimerase Dirigida por RNA , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A method is presented for improved detection of pseudouridine in nucleoside mixtures based on the specific derivatization with methyl vinyl sulfone followed by analysis by capillary HPLC-mass spectrometry. Reaction conditions were optimized in order to obtain the best yield and specificity. The method was successfully applied to different nucleoside mixtures.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Pseudouridina/análise , Sulfonas/química , Cromatografia Líquida de Alta Pressão/instrumentação , Espectrometria de Massas , Pseudouridina/química , Sensibilidade e EspecificidadeRESUMO
tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.
