Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopyReport as inadecuate




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Journal of Biomolecular NMR

, Volume 51, Issue 4, pp 505–517

First Online: 30 October 2011Received: 18 July 2011Accepted: 11 October 2011

Abstract

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 C-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-C-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-C-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.

KeywordsSite selective labeling DL323 Pyruvate-3 Ribose and nucleobase RNA Structure and dynamics AbbreviationsAMPAdenosine 5′-monophosphate

CMPCytidine 5′-monophosphate

UMPUridine 5′-monophosphate

GMPGuanosine 5′-monophosphate

R5PRibose-5-phosphate

FBPFructose-6-bisphosphate

F6PFructose-6-phosphate

GA3PGlyceraldehyde-3-phosphate

G6PDHGlucose-6-phosphate dehydrogenase

K10zwfGlucose-6-phosphate dehydrogenase mutant

GlyGlycine

SerSerine

noPPPNon-oxidative pentose phosphate pathway

OAAOxaloacetate

DHAPDihydroxyacetone phosphate

oPPPOxidative pentose phosphate pathway

rNTPsRibonucleoside triphosphates

TIMTriosephosphate isomerase

PEPPhosphoenolpyruvate

G6PGlucose-6-phosphate

Ru5PRibulose-5-phosphate

X5PXylulose-5-phosphate

R5PRibose-5-phosphate

S7PSedoheptulose-7-phosphate

E4PErythrose-4-phosphate

3PG3-Phosphoglycerate

6PG6-Phosphogluconate

6PGA6-Phosphogluconate-δ-lactone

Electronic supplementary materialThe online version of this article doi:10.1007-s10858-011-9581-6 contains supplementary material, which is available to authorized users.

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Author: Chandar S. Thakur - T. Kwaku Dayie

Source: https://link.springer.com/







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