Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 140

Fig. 8.1 1 H 1D spectrum of a 32 nt RNA (PDB ID 1XHP). Characteristic 1 H chemical shift ranges
are indicated for nitrogen-bonded protons ( white bars ) and carbon-bonded aromatic and ribose
protons ( black bars ). Imino protons involved in Watson-Crick (WC) base-pairing have a chemical
shift range of 10-15 ppm. DSS is 4,4-dimethyl-4-silapentane-1-sulfonic acid, a chemical shift cali-
bration standard
(Tzakos et al. 2006 ; Fernández and Wider 2003 ). TROSY partially counters the
dipole-dipole relaxation between neighboring nuclei using constructive interfer-
ence from chemical shift anisotropy (Hennig et al. 2001 ; Pervushin et al. 1997 ) .
Currently, the practical size limitation for RNA structure determination by NMR is
approximately 40 kDa, or around 100 nucleotides. However, in some favorable
cases, NMR can be used to determine secondary structures of much larger RNAs
(Lu et al. 2011 ). Approaches for resolving chemical shift overlap for large RNAs
are discussed in greater detail in Sect. 8.1.2.3 . The second potentially limiting fac-
tor is sample concentration. NMR is an inherently insensitive technique, so the
method generally requires high concentrations of sample. For biological macro-
molecules, this is typically between 0.1 and 2 mM. Even with 1 mM sample con-
centrations, signal averaging of multiple experiments is typically used to increase
the signal-to-noise.
Resolution is the third major challenge in NMR and is particularly problematic
for RNA. Unlike proteins, which are composed of 20 distinct amino acids, nucleic
acids are made up of only four chemically similar nucleotides, which results in a
small degree of chemical shift dispersion (Fig. 8.1 ). Additionally, the proton density
within a nucleotide is concentrated primarily in the ribose ring, and the majority of