Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 147

can be measured by time-resolved NMR methods (Furtig et al. 2007 ; Schanda et al.
2005 ; Lee et al. 2010 ) .
The study of RNA dynamics by NMR has been expanded by the measurement
and analysis of RDCs. RDCs report changes in bond orientation relative to the mag-
netic field resulting from internal and overall molecular motions (Tolman 2001 )
(Fig. 8.4 ). These couplings report on the weighted average of all conformations
sampled by a macromolecule over the course of picoseconds to milliseconds. This
time range encompasses both local motions as well as large-scale conformation
changes associated with RNA domain motions (Fig. 8.6 ).
A-form helices are the predominate building blocks of RNA structures. Because
they are semi-rigid and highly regular, their overall geometries are independent of
sequence as long as base-pairing is maintained (Wang et al. 2010 ) . Therefore, a
large, complex RNA structure can be thought of as a combination of A-form heli-
cal domains, which can be directly linked or separated by single-stranded regions
that form loops, turns, bulges, and linkers (Hermann and Patel 2000 ; Butcher and
Pyle 2011 ). These elements allow RNA to sample a wide range of conformations.
NMR provides a unique way to quantitatively characterize domain motion and
orientation using RDCs (Getz et al. 2007 ; Zhang and Al-Hashimi 2009 ) . Domain
motion and orientation can be efficiently examined using RDCs by solving the
order tensor solution (Bailor et al. 2007 ), which describes the average orientation
of each domain relative to the magnetic field and provides information on direction
and amplitude of motion.
Investigation of RNA dynamics using order tensor analysis is efficient because
solution structures and complete resonance assignments are not required. Order
tensor analysis requires a minimum of five nonparallel RDCs for each helical
domain (excluding RDCs from terminal base-pairs, which are dynamic due to
fraying). Because helices are highly regular structures, they can be accurately
modeled using RNA modeling programs (Bailor et al. 2007 ). Practically, a set of
³11 RDCs is needed for a well-determined order tensor solution. Consider an
RNA molecule with two helices separated by an asymmetrical bulge (Bailor et al.
2007 ). To determine the interhelical bend angle and dynamics between the two
helical domains, order tensor solutions are determined for each helix indepen-
dently. A 13 C, 15 N labeled RNA sample is used to measure aromatic (CH), ribose
(C1¢ H1 ¢ , C2 ¢ H2 ¢ , C3 ¢ H3 ¢, and C4¢ H4 ¢), and imino (NH) dipolar couplings in iso-
tropic and partially aligned conditions. The difference in dipolar coupling in the
partially aligned and isotropic conditions yields the RDC measurement. The dipo-
lar coupling is manifested as a measurable peak splitting (Hz), which should be
measured in duplicate along the 13 C/ 15 N and 1 H dimensions to assess the RDC
error. The order tensor solution for each helix can be computed using software
such as RAMAH (Hansen and Al-Hashimi 2006 ). The Euler angles from the order
tensor solution are used to rotate model A-form helices into their principle axis
system (PAS). In their PAS, the helices are connected according to their covalent
bonding and the interhelical bend angle can be calculated using the Euler-RNA
software (Bailor et al. 2007 ). RNA dynamics are encoded by the general degree of
order (GDO) and h terms in the order tensor solution (Getz et al. 2007 ) . The GDO