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Journal of Biomolecular NMR

, Volume 48, Issue 4, pp 179–192

First Online: 06 November 2010Received: 20 May 2010Accepted: 28 July 2010

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 C-2-glycerol and C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ 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 C-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 C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position ~90% that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ 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.

KeywordsAlternate-site specific labeling CH2-methylene TROSY Ribose and nucleobase RNA Structure and dynamics AbbreviationsAMPAdenosine 5′-monophosphate

CMPCytidine 5′-monophosphate

DHAPDihydroxyacetone phosphate

FBPFructose-6-bisphosphate

F6PFructose-6-phosphate

GA3PGlyceraldehyde-3-phosphate

GlyGlycine

GMPGuanosine 5′-monophosphate

noPPPNon-oxidative pentose phosphate pathway

OAAOxaloacetate

oPPPOxidative pentose phosphate pathway

R5PRibose-5-phosphate

rNMPsRibonucleoside monophosphates

rNTPsRibonucleoside triphosphates

SerSerine

TIMTriosephosphate isomerase

UMPUridine 5′-monophosphate

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Autor: Chandar S. Thakur - Jacob N. Sama - Melantha E. Jackson - Bin Chen - T. Kwaku Dayie

Fuente: https://link.springer.com/







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