Substrate specificity of the pseudouridine synthase RluD
in Escherichia coli
Margus Leppik, Lauri Peil, Kalle Kipper, Aivar Liiv and Jaanus Remme
Institute of Molecular and Cell Biology, Tartu University, Tartu, Estonia
Pseudouridines (Y) are the most common modifications
in stable RNAs. Pseudouridine was discovered as a fifth
nucleotide in yeast tRNA 50 years ago [1]. Pseudo-
uridines are synthesized from uridine by pseudouridine
synthases, a reaction that does not need additional
cofactors or external energy sources. Pseudouridine
synthases are classified into five families according to
their amino acid sequence [2,3]. Despite low sequence
homology of the enzymes, structural comparison of
crystal structures reveals that all pseudouridine synth-
ases share a core with a common fold and a conserved
active site cleft [4].
Pseudouridines are found in all tRNAs and high-
molecular rRNAs. 16S ribosomal RNA from Escheri-
chia coli contains one pseudouridine Y516 formed by
RsuA [5]. 23S rRNA from E. coli contains ten Y resi-
dues, which are made by six enzymes RluA–RluF [6].
Enzymes such as RsuA and RluB isomerize only one
uridine in the substrate RNA whereas others (RluC
and RluD) make three pseudouridines [7–9]. RluA
modifies uridine 746 in 23S rRNA and uridine 32 in
some specific tRNA species [10].
RluD isomerizes uridines at positions 1911, 1915,
and 1917 in stem-loop 69 (H69) of 23S rRNA [8,9].
Y1917 is found at the corresponding position of the
large ribosomal subunit RNAs throughout all king-
doms. It is the most conserved modification in

tions 1911, 1915, and 1917, regardless of the presence of uridine at other
positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of
one U by C has no effect on the conversion of others (i.e. formation of
pseudouridines at positions 1911, 1915, and 1917 are independent of each
other); (c) A1916 is the only position in the loop of helix 69, where muta-
tions affect the RluD specific pseudouridine formation. Pseudouridines
were determined in the ribosomal particles from a ribosomal large subunit
defective strain (RNA helicase DeaD
–
). An absence of pseudouridines in
the assembly precursor particles suggests that RluD directed isomerization
of uridines occurs as a late step during the assembly of the large ribosomal
subunit.
Abbreviations
Y, pseudouridine; ASL, anticodon stem loop; H69, stem-loop 69.
FEBS Journal 274 (2007) 5759–5766 ª 2007 The Authors Journal compilation ª 2007 FEBS 5759
thermodynamic studies on the isolated helix-loop 69
[17].
Deletion of the yfiI(rluD) gene reduces the growth
rate by three- to five-fold [8,9] and leads to accumula-
tion of the precursor particles for the large and small
subunits [18]. Ribosomes lacking RluD specific
pseudouridines are less stable at low magnesium ion
concentration and exhibit reduced activity during
poly(U) translation in vitro [19]. These effects were
attributed to the lack of pseudouridines in H69 [19].
However, in the presence of an as yet unidentified sec-
ond site mutation, bacteria lacking RluD are able to
grow at a similar rate as wild-type cells. This pseudo-
revertant strain does not contain Y in H69 and exhib-

rRNA variants were expressed in vivo and purified by
affinity tag. Pseudouridines around helix-loop 69 were
determined by chemical modification.
Results
RluD synthesizes
Y
only at U1911, U1915, and
U1917
Helix-loop 69 of E. coli 23S rRNA contains uridine at
positions 1911, 1915, and 1917, which are all converted
to pseudouridines by the pseudouridine synthase RluD
(Fig. 1). To test whether or not RluD is able to modify
uridine at other positions of the H69, nucleotides
A1912, C1914, A1916, and A1919 were substituted by
uridine as single point mutations. Mutant genes were
expressed in vivo and the mutant ribosomal particles
were isolated as described in the Experimental proce-
dures. All 23S rRNA variants were incorporated into
fractions 50S and 70S. Pseudouridines were determined
by chemical modification, followed by reverse trans-
criptase directed primer extension. The primer exten-
sion stop on the CMCT treated RNA (+ lane)
indicated the presence of pseudouridine at the particu-
lar position when the stop was not present in the con-
trol (– lane). m
3
Y present at position 1915 [11] causes
primer extension stop independent of CMCT treatment
(Fig. 2). It must be noted that m
3

the wild-type positions (1911 and 1917) and not at
mutant positions. The 23S rRNA variant A1916U
derived from free 50S subunits did not exhibit pseudo-
uridine specific stop sites at positions U1911, U1916,
and U1917 (Fig. 2). Thus, pseudouridine was not
detected in H69 of the 23S rRNA variant A1916U iso-
lated from free 50S subunits. In the 70S particles, the
CMCT induced stop sites were just on the border of
detection limit, indicating that only traces of pseudo-
uridines were present (Fig. 2). This result suggests that
A1916 is an important specificity determinant for
RluD. Pseudouridines were found at wild-type posi-
tions (1911 and 1917) in spite of the presence of uri-
dine at other positions. We conclude that RluD is
specifically recruited to positions U1911 and U1917 of
E. coli 23S rRNA, at least in vivo.
Mutations at position A1916 affect the specificity
of RluD
Substitution of uridine by cytidine at position 1911
leads to the disappearance of a CMCT dependent stop
signal at position 1911 as expected (Fig. 3). Similar
results were obtained with the transition at posi-
tions 1915 and 1917. These mutations had an effect on
the formation of pseudouridine exclusively at the
mutant position and did not affect either of the other
two positions (Fig. 3). Y1917 was also found in the
23S rRNA variant containing the double mutation
U1911C ⁄ U1915C (Fig. 3). Thus, isomerization of uri-
dines 1911 and 1917 in H69 occurs autonomously of
each other and is independent of modification at 1915.

transition of A1916 to G. The effects of the mutations
in 23S rRNA on the pseudouridine formation in H69
are summarized in Table 1. It is evident that only
mutations at position A1916 to G and U affect RluD
activity. A weak stop site at position 1915 was detected
in CMCT untreated samples of 23S rRNA variants
A1916U and A1916G (only 50S fraction), suggesting a
low level of N
3
methylation of uridine.
RluD modification occurs during late assembly
We have analyzed whether RluD forms pseudouridines
during early assembly on naked 23S rRNA or alterna-
tively requires the presence of r-proteins for its activ-
ity. We used an E. coli strain (deaD
–
), negative for the
RNA helicase DeaD (CsdA), which has been shown to
be deficient in ribosomal large subunit assembly [22].
40S particles accumulating in this strain are assembly
precursors of 50S subunits (L. Peil & J. Remme,
unpublished results). We have analyzed 23S rRNA
from 40S, 50S and 70S particles regarding Y residues
in H69 of 23S rRNA. Primer extension analysis
showed that, in the wild-type strain, both 70S and 50S
particles contain RluD specific pseudouridines. 70S
ribosomes from the deaD
–
strain contain Y residues at
positions 1911 and 1917, indicating that RluD is active

A1919U + + ND ND
Fig. 5. Identification of pseudouridines in the helix-loop 69 in differ-
ent stages of 50S biogenesis by primer extension. Ribosomal parti-
cles were isolated from wild-type and the deaD
–
strain. 40S
particles are assembly precursors accumulating in the deaD
–
strain.
RluD specificity in vivo M. Leppik et al.
5762 FEBS Journal 274 (2007) 5759–5766 ª 2007 The Authors Journal compilation ª 2007 FEBS
results). We conclude that RluD directed isomerisarion
of uridines in the helix-loop 69 of 23S rRNA occurs as
a late event during assembly of the ribosomal large
subunit, but before the 50S subunit enters the 70S
pool.
Discussion
Each pseudouridine in eubacterial rRNA is formed by
a single pseudouridine synthase [6]. On the other hand,
some pseudouridine synthases are able to isomerize
several uridines (e.g. RluC and RluD isomerize three
uridines each). It was proposed that RluD recognizes
all uridines in or near the loop of helix 69 and con-
verts them to pseudouridines [6]. This type of regional
specificity was recently found to be used by TruA
which converts any uridine at positions 38–40 of sub-
strate tRNA to pseudouridine [23]. We have tested
whether or not RluD has similar regional specificity by
mutating nucleotides at positions A1912, A1913,
C1914, A1916, and A1919 of H69 to uridine. None of

agreement with the results described in the present
study.
Mutation A1916 to G and U had severe effects on
RluD in vivo. Y1911 and Y1917 were found on the
23S rRNA variants in the 70S ribosomes, albeit at a
reduced level, but not in the 50S fraction. Substitution
of A1916 by U had stronger effect on the RluD com-
pared to A1916G mutation. Thus, the nucleotide
A1916 in 23S rRNA is an important identity element
for pseudouridine synthesis at both positions 1911 and
1917. This indicates that the identity determinants are
at least partially overlapping for both positions.
Although RluD is highly specific to positions 1911 and
1917, this enzyme is insensitive to the base substitu-
tions in the loop of helix 69. This apparent contradic-
tion can be resolved assuming that A1916 is important
for the initial docking of the RluD. Identity determi-
nants required for the pseudouridine formation may lie
within the flipped out conformation of H69 because
base flipping is obligatory for Y synthesis [4].
The cocrystal structure of E. coli TruB bound to the
T-stem and loop fragment of tRNA has been deter-
mined [25]. This structure suggests that TruB recog-
nizes the T-loop by shape and makes sequence specific
contacts with a few invariant nucleotides, such as C56
[25]. Genetic and biochemical data have shown that
isomerization of U55 in tRNA by E. coli TruB or by
its yeast (Saccharomyces cerevisiae) ortholog PUS4 is
sensitive to base substitution in the TY-loop [20,26]. A
second cocrystal structure of pseudouridine synthase

Ribosomal subunit assembly involves folding of
rRNA and r-proteins and association of both into
functional subunits. In addition, post-transcriptional
modifications are made in rRNA and post-transla-
tional modifications added to r-proteins during ribo-
some assembly. Ribosomal 50S subunits are formed
in vivo during 1–2 min after transcription of 23S
rRNA, but an additional 3 min are required before the
large subunits enter the functional 70S pool [28]. This
additional time is probably used for making late
assembly specific modifications of rRNA and r-pro-
teins and for fine adjustment of ribosome structure.
Analysis of the pseudouridylation pattern in ribosome
assembly precursor particles of 23S rRNA has shown
that the pseudouridines in H69 are formed by RluD
during late assembly. RluD can function in ribosome
assembly by helping to refold H69 into a functional
structure. The refolded H69, containing pseudouri-
dines, supports ribosome subunit association. The
results indicating that Y1911 and Y1917 are present in
the 70S ribosomes but not in the 50S subunits on the
23S rRNA variant A1916G are in agreement with the
role of RluD in functional 50S formation. Taken
together, the results suggest that rRNA modification,
in particular by RluD, is an important event during
late assembly of 50S particles.
Experimental procedures
Plasmids and strains
23S rRNA single point mutations were initially constructed
by PCR mutagenesis on plasmid pXB containing the E. coli

lined). The resulting 1123 bp PCR product was gel-purified
using UltraClean 15 DNA Purification kit (MoBio, West
Carlsbad, CA, USA). Twenty nanograms of of purified
PCR product was then electroporated into
MG1655 ⁄ pKD46 competent cells, previously grown in the
presence of 10 mm arabinose and made competent by con-
centrating ten-fold and washing five times with ice-cold
10% glycerol. Selection for the recombination event and
the elimination of pKD46 plasmid was performed as
described previously [31]. Colonies were tested for the rluD
deletion by PCR, using primers flanking the gene, and by
Southern analysis. The rluD::cat strain had a full deletion
of both rluD and yfiH, together with their annotated pro-
moter sequences. The inserted cat cassette had a distance of
90 nt from the b2595 gene and 177 nt from the clpB gene.
Preparation of ribosomes and rRNA
23S rRNA variants A1912U, A1912C, A1913G, C1914U,
A1916G, A1916C, A1916U, A1917G, A1919C, and
A19191U were expressed in the E. coli strain XL1-Blue
transformed with the corresponding ptBBtag plasmid. Cells
were grown at 37 °Cin2· YT medium supplemented with
ampicillin (100 lgÆmL
)1
). Ribosomes were isolated from
cells 2 h after induction with isopropyl thio-b-d-galactoside
(Fermentas, Vilnius, Lithuania) (1 mm) until an attenuance
of 0.2 at D
600
was reached. Other strains were grown in
2 · YT medium until an attenuance of 0.2–0.3 at D

Cl, 1 mm MgOAc, 6 mm b-mer-
captoethanol]. Plasmid encoded ribosomes were eluted in
the same buffer containing 100 m m biotine. Mutant ribo-
somes contained less than 10% wild-type ribosomes accord-
ing to primer extension around the tag site.
23S rRNA variants U1911C, C1914A, U1915C, and
A1918G were expressed in E. coli strain MC315 (Dlac,
DrecA, D7 prrn) [32,33] transformed with pKK3535 deriva-
tives. 70S, 50S, and 30S gradient fractions were collected
and precipitated with 2.5 volumes of ice-cold ethanol.
rRNA was prepared using a modified protocol [12]. In
brief, ribosomes were dissolved in 200 lL of water and
1 mL of PN solution (catalog no. 19071; Qiagen, Hilden,
Germany) was added. Ribosomal proteins were extracted
by vigorous shaking for 20 min at room temperature.
Twenty microlitres of a 50% silica suspension in water was
added and RNA was bound for additional 10 min at room
temperature with gentle mixing. Silica was pelleted by cen-
trifugation at 3000 g for 30 s and washed twice with 70%
ethanol. RNA was eluted with 50 lL of water (10 min at
room temperature).
Determination of pseudouridines
Pseudouridines were determined according to the method
of Ofengand [34]. Fifteen micrograms rRNA were dissolved
in 20 lL of water, 80 lL of BEU buffer (7 m urea, 4 mm
EDTA, 50 mm Bicine ⁄ NaOH pH 8.5) and 20 lLof
CMCT ⁄ BEU buffer (1 m CMCT in BEU buffer) (CMCT;
Sigma-Aldrich Chemie GmbH, Steinheim, Germany) were
added. One hundred microlitres of BEU buffer were added
to 15 lg of rRNA in 20 lL of water serving as negative

Acknowledgements
This paper is dedicated to the memory of Professor
James Ofengand from the Univeristy of Miami. We
thank Dr U
¨
. Maiva
¨
li for critically reading the manu-
script and Joachim Gerhold (both from Tartu Univer-
sity) for correcting the English. The research was
supported by Estonian Science Foundation Grants
No. 5822 (JR) and No. Aivar (AL).
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