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Analysis of Ribose Ring Dynamics in RNA Molecules Using 13C NMR
Author(s) -
Johnson James Edward,
Julien Kristine R.,
Hoogstraten Charles G.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a69-d
Subject(s) - ribose , residual dipolar coupling , chemistry , nucleotide , rna , ring (chemistry) , molecular dynamics , ribozyme , molecule , carbon 13 nmr , nuclear magnetic resonance spectroscopy , stereochemistry , computational chemistry , biochemistry , organic chemistry , gene , enzyme
Structure‐function relationships of catalytic RNA molecules are important in understanding their mechanisms of catalysis. Nuclear magnetic resonance (NMR) 13 C spin relaxation experiments are useful tools to study dynamic motions of these molecules. However, these experiments thus far have been limited largely to the nucleotide base due to 13 C‐ 13 C dipolar and scalar coupling within the ribose ring. In the lead‐dependent ribozyme, spin relaxation analysis of the nucleotide bases led to the model that the nucleotide base of Cyt‐6 flips out of the helical structure to form the active conformer. We developed a novel isotope labeling scheme whereby the ribose ring has an alternating 13 C‐ 12 C pattern allowing 13 C relaxation experiments to be extended to the ribose ring. This was achieved by using mutant E. coli cells grown with selective 13 C glycerol as the sole carbon source. Using AMP with our specific labeling pattern compared with uniformly labeled AMP solvated in glycerol, we have determined T 1 and T 1 ρ relaxation rates. We show that the 13 C‐ 13 C interactions lead to errors in both T 1 and T 1 ρ rates, and that the use of our labeling pattern alleviates these errors. Finally, we have incorporated our isotopic labeling scheme into RNA molecules including the lead‐dependent ribozyme. We tested a mechanistic hypothesis including correlated motions of the ribose sugar pucker with previously observed nucleotide base dynamics. From all the results we have obtained, we are confident that 13 C NMR spin relaxation techniques can be expanded to the ribose ring to help understand the role of molecular motions in RNA function. Supported by NIH grant RO1 GM069742.