Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Abstract

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs.