Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 142

Fig. 8.3 The HNN-COSY experiment directly detects base-pairing by correlating the acceptor and
donor nitrogen atoms involved in the hydrogen bond. Magnetization is transferred from the imino
proton to the donor nitrogen (1), and then from the donor nitrogen through the hydrogen bond to the
acceptor nitrogen (2). After labeling the magnetization, it is transferred back to the imino proton
through the reverse process. Adapted from Tzakos et al. ( 2006 )

hydrogen bonding. The simplest application of this experiment correlates the
donor and acceptor 15 N atoms across N-H hydrogen bonds in Watson-Crick

base-pairs (Tzakos et al. 2006 ; Dingley and Grzesiek 1998 ) . During the experiment,
magnetization is transferred in two steps (Fig. 8.3 ): first, from the imino proton
to its directly bonded donor nitrogen, and second, from the donor nitrogen
through the hydrogen bond to the acceptor nitrogen. The magnetization is trans-
ferred back to the imino proton through the reverse of the first two steps. These
experiments have been extended to detect NH

N

O = C , OH N- , OH O = P and

hydrogen bonds (Duchardt-Ferner et al. 2011 ) .

NH O = P

2

8.1.2.2 Towards Solving NMR Structures

The quality of the ensemble of NMR structures is dependent upon the quality of
restraints, which includes the overall number of distance restraints (NOEs) mea-
sured (Allain and Varani 1997 ). RNA structure determination by NMR has histori-
cally relied on the measurement of many short range distance restraints. This
approach requires unambiguous chemical shift assignment for as many of the pro-
tons in the RNA as possible. For RNAs larger than 30-40 nucleotides, uniform 13 C,
15 N labeling may not be sufficient to resolve proton spectral overlap. To circumvent
this problem, RNAs can be labeled using nucleotide-type specific labeling strategies
(Hennig et al. 2001 ; Lu et al. 2010 ). Selective deuteration (Davis et al. 2005 ) and
perdeuteration (Lu et al. 2010 ) can also be used to dramatically simplify NMR spec-
tra and resolve chemical shift overlap (discussed in Sect. 8.1.2.3 ).
Chemical shift assignment is accomplished in two basic ways. The first and most
direct is via through-bond experiments that take advantage of scalar couplings
between protons and neighboring 1 H, 13 C, and 15 N nuclei (Hart et al. 2008 ; Hennig
et al. 2001 ; Tzakos et al. 2006 ; Clos et al. 2011 ). Because chemical shifts are domi-
nated by local electronic environment, the chemical shift of the proton and its