Investigating RNAs Involved in Translational Control by NMR and SAXS - Biophysical Approaches to Translational Control of Gene Expression - page 145

b

a

6.0

6.0 5.8 5.6 5.4 5.8 5.6 5.4

(24)

4.0 4.0

43

15

25

4.2 4.2

20

34

40

41

14 31

42

26

13

28

12
29

(39)

4.4

4.4

9

4

32

19 30

18

3

16

11

33

4.6

4.6

17

10

35

2

5

27

(1) (8)

6.0 5.8 5.6 5.4 6.0 5.8 5.6 5.4

ω 2 - 1 H (ppm)

ω 2 - 1 H (ppm)

Fig. 8.5 Selective deuteration greatly simplifies NMR spectra (Davis et al. 2005 ) . ( a ) A 900 MHz
2D NOESY spectrum of the inter- and intra-ribose region. ( b ) A 900 MHz 2D NOESY of the same
region of the selectively deuterated sample. A sequential internucleotide walk from H2¢ ( i ) to H1¢
( i + 1) is drawn. Numbers in parentheses are for those sequential peaks not in the region displayed.
Reprinted from Davis et al. ( 2005 ) , with permission from Elsevier

Several labeling strategies can be used to facilitate chemical shift assignment.
Because 2 H nuclei are not visible by 1 H NMR, selective deuteration can greatly
simplify highly crowded regions of the spectrum, like the ribose region (Fig. 8.5 )
(Davis et al. 2005 ). Alternatively, incorporation of a protonated nucleotide-type into
an otherwise perdeuterated RNA chain is another successful strategy for greatly
simplifying NMR spectra of large RNAs (Lu et al. 2010 ; D'Souza et al. 2004 ;
Miyazaki et al. 2010 ). Deuteration also improves relaxation properties for larger
RNAs, leading to an improved signal-to-noise ratio (Scott et al. 2000 ) . Deuteration
has been useful for several large RNA structures (Davis et al. 2005 ; D'Souza et al.
2004 ; Miyazaki et al. 2010 ) .
A second method facilitating chemical shift assignment uses selective nucle-
otide-type 13 C, 15 N labeling in conjunction with filter-edited NOESY experiments
(D'Souza et al. 2004 ; Peterson et al. 2004 ) . Filter-edited NOESY experiments iden-
tify intermolecular NOEs between isotope labeled and unlabeled nucleotides. We
found that 2D filter-edited experiments take approximately 24 h and were sufficient
for assignment of an 86 nt RNA complex (Davis et al. 2005 ) .

8.1.2.4 RNA Dynamics

It is increasingly clear that RNA can sample a wide range of structural conformations
due to inherent dynamics (Bailor et al. 2010 ). A complete understanding of RNA
structure and function requires knowledge of its dynamic motions. Many biophysical