Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

the past, such scalar couplings have hindered the interpretation of relaxation dispersion data (89, 90) . Using the bacterial A-site RNA as a model system, we were able to capture motions on the microsecond timescale using CPMG experiments to monitor exchange of the ribose C2 residues. It is widely accepted that motions in residues A1492 and A1493 are involved in the discrimination between cognate and near-cognate tRNAs (7) (8) (91) (92) (93) . Most notably, A1493, a residue that flips in and out of the bulge region of A-site showed characteristic dispersion profiles ( Figure 4B ). The extracted k ex and p b values of 3800 ± 200 s −1 and 1.8 ± 0.1% match well the previously reported values of 4000 s −1 and 2.5% determined by relaxation dispersion measurements on the C1 positions of the ribose moieties (8) . Thus, our labels can be used to readily and straightforwardly capture lowly populated states in RNA.