Preparation of Yeast tRNA Sample for NMR Spectroscopy.
In vitro transcription
In vivo RNA production
NMR
NMR spectroscopy
Post-transcriptional modifications
RNA purification
Transfer RNA
tRNA
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
20 Jun 2020
20 Jun 2020
Historique:
received:
30
01
2020
revised:
20
04
2020
accepted:
23
04
2020
entrez:
4
3
2021
pubmed:
5
3
2021
medline:
5
3
2021
Statut:
epublish
Résumé
Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the maturation of a model yeast tRNA with time-resolved NMR, we showed that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. The implementation of this method requires the production for NMR spectroscopy of tRNA samples with different modification status, in order to identify the NMR signature of individual modifications. The production of tRNA samples for the analysis of modification pathways with NMR spectroscopy will be presented here and examplified on the yeast tRNA
Identifiants
pubmed: 33659318
doi: 10.21769/BioProtoc.3646
pii: e3646
pmc: PMC7842793
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e3646Informations de copyright
Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare no competing interest.
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