Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli
 

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle


Abstract Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3â?² and C5â?² carbon positions. Consequently the C1â?², C2â?² and C4â?² positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4â?² carbon position, such that the C2â?² and C3â?² positions suffer from multiplet splitting but the C5â?² position remains singlet and the C1â?² position shows a small amount of residual C1â?²â??C2â?² coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5â?² position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4â?² carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems.

• Content Type Journal Article
• DOI 10.1007/s10858-010-9454-4
• Authors
  • Chandar S. Thakur, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
  • Jacob N. Sama, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
  • Melantha E. Jackson, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
  • Bin Chen, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA
  • T. Kwaku Dayie, Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), College Park, MD 20742-3360, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR