Supplementary Figures
Figure S1. ITC NC titration data obtained for rUAUCUG. Representative ITC data
for MLV NC binding to rUAUCUG. Top: Raw data. Bottom: Fit (solid line) to
baseline corrected ITC data, affording a dissociation constant of 75 ± 12 nM (reported as
the mean ± standard deviation from three experiments).
Figure S2. NOESY data obtained for the NC-mCES complex. Left: Overlayed
portions of 2D NOESY data obtained for NC-mCES samples containing a ~30% excess
of mCES, and with perdeuterated/selectively protonated-cyctidine (black) and –uridine
(red). f and b refer to signals associated with the free and bound RNA, respectively.
“Intermolecular” cross peaks associated with the free RNA signals result from chemical
exchange between free and bound RNA, and provided redundant assignments that were
particularly helpful for signals that were exchange-broadened by NC binding. Right:
Portions of 3D 13C-edited NOESY data obtained for an NC-mCES sample containing
15
N,13C-labeled NC, showing intermolecular cross peaks associated with Lys 30, Ala 36
and Ala 27. These data enabled assignment of the protein components of the
intermolecular NOES observed in the 2D NOESY spectra.
Figure S3. 2D NOESY data obtained for the NC-r(UAUCUG) complex. Downfield
(left) and upfield (right) portions of 2D NOESY data obtained for the NC-r(UAUCUG)
complex (800 MHz, T=15 °C, pH 7.0). Intermolecular NOEs are denoted by dashed
lines. Chemical shifts and NOE cross peak patterns were very similar to those observed
in the NMR data obtained for the NC-mCES complex (see Supplementary Figure S2).
Figure S4. NMR data obtained for DIS-2 constructs. Portions of 2D NOESY spectra
obtained for mutant (M2, (a) and M1, (b)) and native (c) forms of DIS-2 with sequences
shown in Figures 3c and 1b, respectively. Signals associated with U288 exhibit
significantly different chemical shifts in the alternate monomer conformations, which are
discretely observed in the mutant RNAs and simultaneously observed in the native
construct. (d) Downfield portion of the 2D ROESY spectrum obtained for the 32 residue
native DIS-2 RNA (C278-G309). At the low RNA concentration employed (100 M),
only signals for the monomeric species are observed. Exchange cross peaks correlate
signals of the two alternate hairpin conformations shown in Figure 1b. The letters (a) and
(b) in the figure denote RNA conformations shown on the left (shaded red) and right
(shaded green), respectively, in Figure 1b. At higher RNA concentrations, signals for the
duplex form of DIS-2 are also observed (see ref. 14 for details).
Figure S5. Stereo views of the best fit superpositions of (a) stem loop DIS-2 and the
UCUG linker, (b) stem loop SL-C, and (c) stem loop SL-D from an ensemble of 20
refined NC-mCES structures. The UCUG element is disordered relative to the rest of the
RNA, but exhibits a high degree of internal convergence (see Figure 1e).