Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 160

along a 5-50 °C temperature gradient. Based on the measured imino 1 H exchange
rates, D G, DH, and DS were calculated for each base-pair. This information is
consistent with a zipper-type of unfolding mechanism for this RNA thermometer
(Rinnenthal et al. 2010 ). Surprisingly, mutations were found to have long-range
effects on base-pair stability over a distance of six base-pairs, an effect which
may be transduced via perturbations of the RNA hydration shell (Rinnenthal
et al. 2010 ) .

8.2.2.4 Riboswitches

Riboswitches typically reside in the untranslated 5¢ UTR regions of bacterial mRNA
(Haller et al. 2011 ). These regulatory RNA elements either increase or decrease
expression of the downstream gene as a result of ligand binding (Serganov and Patel
2007 ). Riboswitches are composed of two structural elements: an aptamer domain,
which binds the ligand (usually a metabolite), and an expression platform, which is
linked to the aptamer domain and undergoes a conformational change in response
to ligand binding. Multiple crystal structures have been solved of various riboswitch
aptamer domains bound to their ligands (Edwards et al. 2007 ; Alexander 2009 ;
Henkin 2008 ). While the crystal structures reveal how ligands are bound, they do
not reveal the dynamic interplay between ligand binding and the resulting confor-
mational change in the expression platform. NMR has proved useful for character-
ization of purine riboswitch folding (Buck et al. 2010 ; Ottink et al. 2007 ; Noeske
et al. 2007a, b ). Recently, the folding of an adenine sensing riboswitch, which regu-
lates synthesis of enzymes responsible for purine synthesis, was followed for 3 min
using time-resolved NMR (Lee et al. 2010 ). Ultrafast acquisition of 1 H- 15 N 2D
HSQC spectra revealed that after the addition of the adenine metabolite and magne-
sium to the free RNA solution, the core ligand-bound structure formed in the first
20 s. Within 30 s of core formation, tertiary interactions between two helical loops
were stabilized; however, stabilization of all anticipated base-pairs lasted up to
3 min. The sensitivity of SAXS to overall molecular shape also renders this tech-
nique appropriate for characterization of riboswitches (Rambo and Tainer 2010a ;
Kulshina et al. 2009 ; Stoddard et al. 2010 ; Baird and Ferré-D'Amaré 2010 ; Lipfert
et al. 2010 ; Baird et al. 2010 ), which may have multiple conformations in their
unbound states.

8.3 Summary

Only 2% of the human genome is translated into protein, whereas more than 80% is
transcribed into RNA (ENCODE Project Consortium 2007 ) . Given the abundance
of RNA in human biology, it is clear that RNA structural studies are going to be
important for many years. As more RNAs are investigated, more functions will be
discovered, including new strategies for regulating translation. Both NMR and