Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 141

protons in the ribose ring resonate in a narrow spectral window of ~1 ppm. This
high level of chemical shift overlap can make it challenging to obtain complete
resonance assignments. Therefore, isotopic 13 C and 15 N labeling (Nikonowicz et al.
1992 ; Nikonowicz and Pardi 1993 ) is required to resolve 1 H chemical shifts into
additional dimensions by correlating 1 H shifts to their bonded carbon and/or nitro-
gens. Such multidimensional heteronuclear NMR experiments are instrumental in
resolving overlapped 1 H chemical shifts (Hennig et al. 2001 ) .

8.1.2.1 NMR Analysis of Base-Pairing Interactions

and Secondary Structure

The first step in investigating RNA structure using NMR is measuring a 1D 1 H spec-
trum. The observation of imino proton resonances from G and U nucleotides (note
there are no imino protons on A and C), in the 10-15 ppm range, is indicative of
hydrogen bond formation and base-pairing (Fig. 8.1 ). The imino resonances can be
assigned to their respective nucleotides using 1 H- 1 H 2D nuclear Overhauser effect
spectroscopy (NOESY) experiments. The NOESY experiment provides informa-
tion about the number and type of base-pairs, as well as their sequential neighbors
(Heus and Pardi 1991 ) . In a 1 H- 1 H 2D NOESY experiment, cross-peaks (NOEs) are
observed between protons that are less than 6 Å apart. In a standard A-form helix,
an NOE cross-peak is observed between adjacent base-pairs (Hennig et al. 2001 )
(Fig. 8.2b ). Thus, sequential neighbors for base-pairs are identified by “walking-
through” these cross-peaks (Fig. 8.2c ).
The HNN-COSY experiment (Dingley and Grzesiek 1998 ) is extremely use-
ful for investigating base-pairing in 15 N-labeled RNA, because it directly detects

Fig. 8.2 1 H- 1 H 2D NOESY experiment for assignment of imino protons in the SIV frameshift
stem-loop RNA structure (Marcheschi et al. 2007 ) . ( a ) RNA secondary structure. ( b ) Base-pairs
stacked in A-form helical regions are oriented such that the G and U imino protons are within 5 Å
and will exhibit a cross-peak in the 1 H- 1 H 2D NOESY. ( c ) Zoomed-in region of 2D NOESY show-
ing imino cross-peaks. A sequential walk is shown. Colored lines correspond to ( a )