Unraveling the structural complexity in a single-stranded RNA tail: implications for efficient ligand binding in the prequeuosine riboswitch

Eichhorn, Catherine D.; Feng, Jun; Suddala, Krishna C.; Walter, Nils G.; Brooks, Charles L.; Al-Hashimi, Hashim M.

NMR chemical shifts are extremely sensitive probes of the local electronic environment for a given bond vector and can provide useful structural information (55) (56) (57) (58) . Highly disordered residues are expected to have chemical shifts similar to nucleotide monophosphates (NMPs). While the chemical shifts of terminal residues are similar to their NMP analogs, increasing differences are observed when approaching the polyadenine core with the greatest differences observed for A30-32 (Supplementary Figure S2 ). The directionality of the chemical shifts is consistent with increased formation of stacking interactions towards the center of the tail (57) . This is further supported by chemical shift perturbations in a trajectory toward the NMPs with increasing temperature (data not shown). Alternatively, addition of magnesium up to 4 mM results in slight chemical shift perturbations farther from NMPs, consistent with previous studies suggesting that increases in ionic strength stabilize ssRNA stacking interactions (59) (data not shown). In contrast polyU has near-identical ( 0.1 ppm) chemical shifts to UMP (Supplementary Figure S2) . Thus, consistent with NOE data, the chemical shift data suggest a comparatively stacked core with a growing level of disorder towards the terminal ends. Normalized resonance intensities (33) further support these observations, which gradually increase towards the terminal ends, consistent with a higher level of pico-to nanosecond motions (Supplementary Figure S2) .