Biosynthetic preparation of 13C/15N-labeled rNTPs for high-resolution NMR studies of RNAs.
High-resolution investigations of the structure and dynamics of RNA molecules by nuclear magnetic resonance (NMR) methodologies require the production of (13)C/(15)N-isotopically labeled samples. A common strategy entails the preparation of (13)C/(15)N-enriched ribonucleoside 5'-triphosphates (rNTPs) to be incorporated into RNA oligomers by in vitro transcription. Here, we describe the methods to obtain isotopically labeled rNTP in a uniform or selective fashion from bacterial cultures, using common and versatile E. coli strains. This chapter also covers procedures for extraction and digestion of the total RNA from bacterial cells, purification of the ribonucleoside 5'-monophosphates and their enzymatic phosphorylation to rNTPs.
Randall Division of Cell and Molecular Biophysics, King's College London, London, UK.
Luigi
L
Martino
Maria R
MR
Conte
D000085:Acetates / Q000494:pharmacology; D002247:Carbon Isotopes / Q000737:chemistry; D049109:Cell Proliferation; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D005561:Formates / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006868:Hydrolysis; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes / Q000737:chemistry; D010766:Phosphorylation; D012313:RNA / Q000096:biosynthesis* / Q000737:chemistry*; D012265:Ribonucleotides / Q000096:biosynthesis* / Q000737:chemistry*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D000085:Acetates; D002247:Carbon Isotopes; D003470:Culture Media; D005561:Formates; D009587:Nitrogen Isotopes; D012265:Ribonucleotides; C030544:formic acid; D012313:RNA; D005947:Glucose; D005990:Glycerol