caa a22 4500 445325380 CHVBK 20180317142718.0 cr unu---uuuuu 170323e20111201xx s 000 0 eng 10.1007/s10858-011-9581-6 doi (NATIONALLICENCE)springer-10.1007/s10858-011-9581-6 Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy [Elektronische Daten] [Chandar Thakur, T. Dayie] Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of 13C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-13C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-13C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides. The Author(s), 2011 Site selective labeling nationallicence DL323 nationallicence Pyruvate-3 nationallicence Ribose and nucleobase nationallicence RNA nationallicence Structure and dynamics nationallicence AMP : Adenosine 5′-monophosphate nationallicence CMP : Cytidine 5′-monophosphate nationallicence UMP : Uridine 5′-monophosphate nationallicence GMP : Guanosine 5′-monophosphate nationallicence R5P : Ribose-5-phosphate nationallicence FBP : Fructose-6-bisphosphate nationallicence F6P : Fructose-6-phosphate nationallicence GA3P : Glyceraldehyde-3-phosphate nationallicence G6PDH : Glucose-6-phosphate dehydrogenase nationallicence K10zwf : Glucose-6-phosphate dehydrogenase mutant nationallicence Gly : Glycine nationallicence Ser : Serine nationallicence noPPP : Non-oxidative pentose phosphate pathway nationallicence OAA : Oxaloacetate nationallicence DHAP : Dihydroxyacetone phosphate nationallicence oPPP : Oxidative pentose phosphate pathway nationallicence rNTPs : Ribonucleoside triphosphates nationallicence TIM : Triosephosphate isomerase nationallicence PEP : Phosphoenolpyruvate nationallicence G6P : Glucose-6-phosphate nationallicence Ru5P : Ribulose-5-phosphate nationallicence X5P : Xylulose-5-phosphate nationallicence R5P : Ribose-5-phosphate nationallicence S7P : Sedoheptulose-7-phosphate nationallicence E4P : Erythrose-4-phosphate nationallicence 3PG : 3-Phosphoglycerate nationallicence 6PG : 6-Phosphogluconate nationallicence 6PGA : 6-Phosphogluconate-δ-lactone nationallicence Thakur Chandar Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), 20742-3360, College Park, MD, USA aut Dayie T. Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), 20742-3360, College Park, MD, USA aut Journal of Biomolecular NMR Springer Netherlands 51/4(2011-12-01), 505-517 0925-2738 51:4<505 2011 51 10858 https://doi.org/10.1007/s10858-011-9581-6 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10858-011-9581-6 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Thakur Chandar Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), 20742-3360, College Park, MD, USA aut NATIONALLICENCE 700 1- Dayie T. Department of Chemistry and Biochemistry, Center for Biomolecular Structure & Organization, University of Maryland, 1115 Biomolecular Sciences Bldg (#296), 20742-3360, College Park, MD, USA aut NATIONALLICENCE 773 0- Journal of Biomolecular NMR Springer Netherlands 51/4(2011-12-01), 505-517 0925-2738 51:4<505 2011 51 10858 Metadata rights reserved Springer special CC-BY-NC licence nationallicence SWISSBIB 445325380 BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer