Pseudouridines in rRNA helix 69 play a role in loop stacking interactions

Article abstract of DOI:10.1039/b812731j

The ¹H NMR spectra of RNAs representing E. coli 23S rRNA helix 69 with [1,3-¹⁵N]pseudouridine modification at specific sites reveal unique roles for pseudouridine in stabilizing base-stacking interactions in the hairpin loop region. The 2008 Royal Society of Chemistry.

Full text of DOI:10.1039/b812731j

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Pseudouridines in rRNA helix 69 play a role in loop stacking interactions†
Jean-Paul Desaulniers,‡ Yu-Cheng Chang,§ Raviprasad Aduri, Sanjaya C. Abeysirigunawardena,
John SantaLucia, Jr. and Christine S. Chow*
Received 24th July 2008, Accepted 1st September 2008
First published as an Advance Article on the web 19th September 2008
DOI: 10.1039/b812731j
The 1H NMR spectra of RNAs representing E. coli 23S rRNA
helix 69 with [1,3-15N]pseudouridine modification at specific
sites reveal unique roles for pseudouridine in stabilizing base-
stacking interactions in the hairpin loop region.
unique stacking interactions and/or structures. Additional imino
proton resonances that are not associated with standard base-
pairing schemes are much more difficult to assign and often involve
complex 2D NMR techniques. Pseudouridine-containing RNAs
(Fig. 1) contain additional imino protons that do not participate in
traditional base-pairing schemes, but give rise to peaks in 1H NMR
spectra. Here, we report on the site-specific incorporation of 15N-
enriched Ws at various positions of hairpin RNAs representing
Helix 69 (H69) of E. coli 23S rRNA is an RNA hairpin motif
located at the heart of the peptidyltransferase center of the
ribosome, which is proposed to have a functional role in protein
translation initiation, translation termination, and/or ribosome
recycling.1 As such, H69 has gained attention as a potential target
for the development of new anti-bacterial, anti-infective agents.
To date, several X-ray and cryo-EM structures have revealed
that H69 adopts significantly different conformations and makes
contacts with RNAs, proteins, and small molecules.1,2 Despite a
number of different static structures, little structural information
exists for H69 within a solution-based environment. One challenge
associated with generating models of H69 for solution studies is
the presence of a highly abundant modified residue known as
pseudouridine.3
1
H69 in order to identify imino resonances within the H NMR
1
spectrum. By doing so, loop W imino proton resonances in H
NMR spectra are assigned unambiguously, thus alleviating the
need for complex 2D techniques.
In this study, H69 RNAs with 15N-labeled W at three different
positions were produced (Fig. 1) by using chemical synthesis.
The generation of these RNAs first required the synthesis of 15N-
labeled W (see ESI†). Previously, routes were developed for [1-15N]-
or [3-15N]-enrichment of W.8 Here, 15N was incorporated at both
the N1 and N3 positions of W in order to examine both imino
1
protons in H NMR experiments. Due to the presence of both
uridine and W residues in H69, enzymatic synthesis with T7 RNA
polymerase could not be used to produce 15N-labeled RNAs.
After 15N-W was generated, it was mixed with a sample of
natural abundance (14N-W) such that the level of 15N enrichment
was 50%. Therefore, any imino proton attached to W that
exchanges slowly with water (N1H and N3H) is observed as a
combination of a singlet and doublet peak. The use of a 1 : 1
mixture of 15N : 14N was essential in the assignment of various
imino peaks, due to the presence of multiple imino peaks within
the region. The mixed-label W phosphoramidite was synthesized
(see ESI†) and used to generate three RNA hairpins representing
modified H69 bearing 15N-labeled Ws at each of the three positions,
1911, 1915, and 1917 (Fig. 1).5d
Among the more than 100 known RNA modifications in nature,
pseudouridine (W) is the most common.4 Three conserved W
residues are present in H69 (Fig. 1) at positions 1911, 1915, and
1917 (E. coli numbering). Pseudouridine plays an important role
in fine tuning the stability of RNA hairpins and other structural
motifs.5 The most significant effect of pseudouridylation is the
introduction of an additional imino group in the pyrimidine ring.
Because of the significance of H69 within the ribosome, a primary
goal of this work was to determine whether these imino protons
contribute to the tertiary structure of H69.
1H NMR spectroscopy is a useful starting point for RNA struc-
ture determination because the imino proton region (9–15 ppm)
is often the simplest portion of an NMR spectrum to interpret.6
Typically, the guanine and uracil imino protons that are involved
in base pairing are observed (90% H2O–10% D2O) and assigned
using 1D NOE difference spectroscopy or novel NMR techniques
with 15N, 13C-labeled RNAs.7 Non-traditional hydrogen-bonding
patterns between imino resonances can also arise in RNA due to
The stem-region proton resonances of H69 were previously
assigned using 1D NOE difference spectroscopy.5d In the case
of pseudouridine, the imino resonances are typically assigned
based on the presence of an NOE from N1H to H6 (and lack of
NOE when NH3 is irradiated). The three new RNAs, 15NWWW,
W15NWW, and WW15NW, show nearly identical 1D 1H NMR
◦
spectra at 21 and 37 C (data shown for 15NWWW in the ESI†).
Department of Chemistry, Wayne State University, Detroit, Michigan,
48202, USA. E-mail: csc@chem.wayne.edu; Fax: 313-577-8822; Tel: 313-
577-2594
† Electronic supplementary information (ESI) available: Synthetic
schemes, experimental details, 1H, 13C, 15N, and MS spectra, RNA
experimental methods, 1D NOE spectra, and temperature-dependent
NMR spectra. See DOI: 10.1039/b812731j
‡ Present address: University of Ontario Institute of Technology, Faculty
of Science, 2000 Simcoe Street North, Oshawa, Ontario, L1H 7K4,
Canada.
§ Present address: The Methodist Hospital Research Institute, Weill
Medical College of Cornell University, 6565 Fannin Street, Houston, Texas
77030, USA.
As the temperature is lowered to 3 ◦C, several peaks become
apparent in the imino region of the spectra (Fig. 2). Since the
level of 15N enrichment in labeled Ws is 50%, the imino protons
(N1H and N3H) are each observed as a combination of a singlet
and doublet peak (these peaks will be referred to as a triplet). For
the 15NWWW RNA, 15N-enrichment of W1911 allows confirmation
of the previously assigned resonances of N1H and N3H at 10.3
and 13.1 ppm, respectively. Irradiation of the 10.3 ppm peak
gives rise to an NOE at 7.0 ppm, assigned to W1911H6. In the
case of W15NWW, peaks at 10.7 and 10.4 ppm, which are broad
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Fig. 1 The structure of pseudouridine (W) and secondary structures of RNA hairpins representing H69 (positions 1906–1924) of E. coli 23S rRNA are
shown. The nitrogens of W are enriched with 15N, and circles indicate [1,3-15N]W positions in the H69 hairpins.
Fig. 2 The 1D imino proton (uridine H3/guanine H1/pseudouridine H1 and H3) NMR spectra at 3 ◦C (500 MHz) of 15NWWW (A), W15NWW
(B), and WW15NW (C) RNAs (90% H2O–10% D2O, 0.5–1 mM) in 30 mM NaCl, 10 mM sodium phosphate, 0.5 mM Na2EDTA, pH 5.8, are shown.
singlets in 15NWWW and unlabeled WWW spectra, are triplets,
indicating that both imino protons of W1915 exchange slowly with
solvent. The resonance at 10.7 ppm is assigned to W1915N1H, based
on the fact that it has a strong NOE to W1915H6 at 7.4 ppm
(see ESI†). Therefore, the 10.4 ppm triplet peak is assigned to
W1915N3H. This result is confirmed by the 15N HMQC spectrum
of W15NWW, which reveals two cross peaks (see ESI†) at 135
(N1) and 159 ppm (N3). These chemical shifts are consistent
with earlier studies in which [1-15N]-W had 15N peaks ∼132–
1
136 ppm and H peaks ∼10.4–10.6 ppm.8a The WW15NW RNA
spectrum has a broad triplet at 11.2 ppm, resulting from 15N
labeling of W1917. Irradiation of this resonance does not show any
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significant NOEs, suggesting that it corresponds to W1917N3H. In
previous NMR studies, resonances between 10.5–10.7 ppm have
been assigned to WN1Hs.5 Thus, the assignment of the 10.4
and 11.2 ppm resonances as loop W1915N3H and W1917N3H is
unique, and suggests a novel structural environment for these
imino protons.
The 1D 1H NMR spectra of 15NWWW reveal slight upfield shifts
(Dppm ∼ 0.1–0.2) of several imino proton peaks as the temperature
is raised from 3 to 37 ◦C (ESI†). In contrast, the imino resonances
of W1915 and W1917 are no longer observed when the temperature
◦
is increased to 15 C. These observations are consistent with the
Fig. 3 A schematic representation of the H69 loop region is shown with
base-stacking interactions between C1914 and W1915 and A1916, W1917, and
H69 loop region undergoing changes in stacking upon heating.
The W imino protons of H69 are clearly observed in the
1H NMR spectra shown in Fig. 2. Typically, imino protons of
RNA loops are not observed, unless they are involved with
base-stacking or hydrogen-bonding interactions, due to rapid
exchange with solvent on the NMR timescale. Of the five W
imino proton resonances, those from W1911N1H and N3H are
the most prominent. This result is consistent with the formation
of an A-W loop-closing base pair through N3H and a putative
water-mediated hydrogen bond between N1H and the phosphate
backbone, which contribute to H69 stability.5d Imino proton
resonances from W1915N1H, W1915N3H, and W1917N3H are also
observed (W1917N1H is the only W imino proton that is not
observed), indicating a specific conformation of the H69 loop
that leads to protection of the W imino protons from solvent
exchange. Previous work by Davis showed that W stacks favorably
with adjacent adenines in a short AAWA oligomer, and the WN1H
(but not N3H) resonance was observed.5b The presence of multiple
adenines in the H69 sequence surrounding W1915 and W1917 suggests
a similar type of stacking interaction as the AAWA tetramer.
However, the W1915 of H69 has unique stacking and/or hydrogen-
bonding interactions, such that both N3H and N1H are protected
from solvent. Although N3H uridine resonances have been
observed by NMR in uridine-containing RNAs,9 the unmodified
H69 construct (containing U1911, U1915, U1917) does not show imino
resonances from any of the N3H uridines.5d Other NMR studies
have shown imino resonances in RNA loop regions, but these
typically arise from non-standard hydrogen-bonding interactions
across loops.10 In the case of H69, there is no evidence for
hydrogen-bonding interactions of Ws with the other loop residues.
Our data reveal that the extra imino protons in the loop
region of the W-containing H69 RNA lead to a structure with
increased base stacking and decreased solvent accessibility. Also
of note is the fact that the unmodified, or uridine, analog of H69
A
1918, and base-pairing interactions between W1911 and A1919.
of the H69 loop participate in significant stacking interactions with
A1916. Evidence for this stacking model is also observed in solution
NMR studies of the WWW RNA 3D structure (see ESI† for a list
of the key NOEs).12 More specifically, the NOE data is consistent
with strong stacking of C1914 with W1915 and tri-nucleotide stacking
with A1916, W1917, and A1918. Such a structure may be important for
fine-tuning the biological function of H69. For example, O’Connor
and Dahlberg showed that mutations in H69 (deletion of A1916 and
insertion of additional adenosines after A1916) cause frame-shifting
and read-through of stop codons.13
The site-specific incorporation of [1,3-15N]-enriched W at
positions 1911, 1915, and 1917 of H69 has allowed for the
unambiguous assignment of the imino proton resonances in the
1D 1H NMR and 1D NOE difference spectra, and determination
of non-Watson–Crick loop interactions that are dependent on
the presence of the modified nucleotide. The 15N-enriched RNAs
will be used for future 15N NMR experiments, including complete
structure analysis of H69, determination of W pKa values in H69,
and monitoring of H69–ligand interactions.
Acknowledgements
We are grateful to Bashar Ksebati and Larry Clos II for helpful
discussions and technical assistance. This work was supported by
the NIH (GM54632).
Notes and references
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