Thông tin tài liệu
Association of RNA with the uracil-DNA-degrading factor
has major conformational effects and is potentially
involved in protein folding
Angela Bekesi
1
, Maria Pukancsik
1
, Peter Haasz
1
, Lilla Felfoldi
1
, Ibolya Leveles
1
, Villo Muha
1
,
Eva Hunyadi-Gulyas
2
, Anna Erdei
3
, Katalin F. Medzihradszky
2,4
and Beata G. Vertessy
1,5
1 Institute of Enzymology, Biological Research Centre, Hungarian Academy of Sciences, Budapest, Hungary
2 Proteomics Research Group, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
3 Department of Immunology, Eo
¨
tvo
¨
s Lora
´
nd University of Sciences, Budapest, Hungary
4 Department of Pharmaceutical Chemistry, University of California, San Francisco, USA
5 Department of Applied Biotechnology, Budapest University of Technology and Economics, Hungary
Introduction
Genomic information stored in DNA is under constant
threat from spontaneous chemical modifications,
occurring under normal physiological conditions. One
of the most frequent spontaneous base transitions
is hydrolytic deamination of cytosine to form uracil
[1–3]. This alteration is mutagenic, as it will convert to
Keywords
conformational states; cotranslational
folding; RNA-assisted folding; RNA binding;
uracil-DNA-degrading factor
Correspondence
A. Bekesi and B. G. Vertessy, Karolina
Street 29, H-1113 Budapest, Hungary
Fax: +36 1 466 5465
Tel: +36 1 279 3116
E-mail: bekesi@enzim.hu;
vertessy@enzim.hu
Website: http://vertessy.enzim.hu
(Received 7 June 2010, revised 29 October
2010, accepted 4 November 2010)
doi:10.1111/j.1742-4658.2010.07951.x
Recently, a novel uracil-DNA-degrading factor protein (UDE) was identi-
fied in Drosophila melanogaster, with homologues only in pupating insects.
Its unique uracil-DNA-degrading activity and a potential domain organiza-
tion pattern have been described. UDE seems to be the first representative
of a new protein family with unique enzyme activity that has a putative
role in insect development. In addition, UDE may also serve as potential
tool in molecular biological applications. Owing to lack of homology with
other proteins with known structure and ⁄ or function, de novo data are
required for a detailed characterization of UDE structure and function.
Here, experimental evidence is provided that recombinant protein is present
in two distinct conformers. One of these contains a significant amount of
RNA strongly bound to the protein, influencing its conformation. Detailed
biophysical characterization of the two distinct conformational states
(termed UDE and RNA–UDE) revealed essential differences. UDE cannot
be converted into RNA–UDE by addition of the same RNA, implying
putatively joint processes of RNA binding and protein folding in this con-
formational species. By real-time PCR and sequencing after random clon-
ing, the bound RNA pool was shown to consist of UDE mRNA and the
two ribosomal RNAs, also suggesting cotranslational RNA-assisted fold-
ing. This finding, on the one hand, might open a way to obtain a conform-
ationally homogeneous UDE preparation, promoting successful
crystallization; on the other hand, it might imply a further molecular func-
tion of the protein. In fact, RNA-dependent complexation of UDE was
also demonstrated in a fruit fly pupal extract, suggesting physiological rele-
vance of RNA binding of this DNA-processing enzyme.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
NLS, nuclear localization signal; UDE, uracil-DNA-degrading factor protein; UDG, uracil-DNA glycosylase.
FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS 295
a stable point mutation (G:C fi A:T). An alternative
pathway for uracil appearance in DNA is thymine-
replacing misincorporation, which is usually efficiently
prevented by the action of dUTPase, which sanitizes
the dUTP ⁄ dTTP pool [4]. Repair of uracil-substituted
DNA is initiated by uracil-DNA glycosylases (UDGs)
in an almost ubiquitous manner. The major UDG
enzyme, the product of the ung gene, excises both
deaminated cytosine and thymine-replacing uracil with
high efficiency. In Drosophila melanogaster, the well-
annotated genome provides unequivocal evidence for
lack of ung [5]. This situation, together with the lack
of dUTPase, which is responsible for preventing
thymine-replacing uracil incorporation into DNA, in
larval tissues may allow accumulation of uracil-
containing DNA at least at specific developmental
stages [6]. By the use of affinity chromatography for
searching additional uracil-DNA-recognizing factors in
late larval extracts, a specific uracil-DNA-degrading
protein (UDE), with homologues only in pupating
insect genomes, was identified and partially character-
ized [7,8].
The DNA-degrading activity of UDE necessarily
involves the formation of a functional DNA–protein
complex. In fact, the DNA-binding ability of UDE
was evident in the first experiments [7], but sequence
homology searches did not reveal any already
described nucleic acid-binding sequence motifs. No 3D
structure is yet available for any UDE-homologue pro-
tein. Trials for structural modelling by fold recognition
methods suggested a novel fold for the UDE protein.
Alignment of UDE homologue sequences identified
five conserved motifs, the first two segments of which
are extended and show significant homology with each
other (termed 1A and 1B) [7]. De novo 3D modelling
of the 1A ⁄ 1B fragments revealed the existence of two
helical bundles with an extended surface formed by
conserved positively charged residues [8]. These results,
together with DNA-binding-induced protection against
limited proteolysis, suggested that DNA binding may
occur in this region [8]. Detailed characterization of
the interaction between UDE and nucleic acids is
expected to provide important insights into specific
aspects of the novel activity.
In the present work, we aimed to characterize
UDE–nucleic acid complexes. Unexpectedly, we found
that a portion of recombinant UDE copurified with
strongly bound RNA. On the basis of this finding, we
decided to investigate the effects of RNA binding on
protein structure and stability in detail. Recombinant
UDE is eluted in two distinct peaks with different
chromatographic techniques, suggesting the existence
of two distinct conformational states of the enzyme:
RNA-bound (RNA–UDE) and RNA-free (UDE).
RNA binding has pronounced effects on thermal sta-
bility and the cooperativity of protein unfolding, as
shown by thermal transitions monitored by CD and
fluorescence spectroscopy. Possible effects of RNA
binding on UDE conformation were assayed by CD
spectroscopy and by titration of hydrophobic surface
patches. The DNA-binding and RNA-binding abilities
of the enzyme forms were measured by electrophoretic
mobility shift assay (EMSA). Characterization of the
bound RNA pool by real-time RT-PCR revealed an
abundance of UDE mRNA as well as of domain V of
23S rRNA. These results, together with the character-
istics of the two distinct conformational states, suggest
putative cofolding of UDE and RNA during transla-
tion. Physiological relevance of RNA–UDE binding
was also demonstrated.
Results
Two distinct molecular populations of UDE can
be separated by Ni
2+
-affinity and size exclusion
chromatography
In a refined purification method with recombinant His-
tagged UDE (see Experimental procedures), gradient
elution was applied on a HisTrap column with AKTA
purifier. Two distinct fractions of intact full-length
UDE eluted at about 300 and 600 mm imidazole,
respectively (Fig. 1A), reflecting their altered affinity
for the Ni
2+
-column. SDS ⁄ PAGE indicated the same
electrophoretic mobility for UDE in both fractions,
arguing against protein degradation or observable
modifications (Fig. 1A, insert panel). MS analysis of
the two protein bands further confirmed the lack of
any detectable post-translational modifications (data
not shown). In the first fraction, UDE eluted together
with a large amount of nucleic acid, whereas the sec-
ond fraction was practically nucleic acid-free. Both
fractions were subjected to size exclusion chromatogra-
phy, where the nucleic acid-containing fraction eluted
in the exclusion volume (corresponding to an apparent
molecular mass larger than 600 kDa), and the nucleic
acid-free fraction eluted at a position corresponding to
an apparent molecular mass of 52 kDa (Fig. 1B). Pre-
viously, we reported purification of UDE by stepwise
Ni
2+
-agarose chromatography, which did not allow
separation of these two fractions [8]. Size exclusion
chromatography of this UDE preparation resulted in
two distinct peaks at the same positions as above. The
observation that full-length UDE can be separated into
two fractions by applying two different chromato-
graphic methods, Ni
2+
-agarose affinity and size
RNA-dependent conformational states of UDE A. Bekesi et al.
296 FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS
exclusion, may suggest the potential existence of two
separate conformational states of the protein, one of
these containing a significant amount of associated
nucleic acid. To address the potential significance of
these conformational states, we first wished to charac-
terize the bound nucleic acid.
Nucleic acid-binding UDE fraction contains RNA,
and not DNA
On native agarose gel, a smeared band was visualized
in the nucleic acid-containing UDE fraction (Fig. 1C).
Test digestions of this UDE preparation by DNase as
well as by RNase A were performed. Surprisingly,
DNase treatment did not significantly perturb the
detected nucleic acid band, whereas RNase treatment
completely eliminated it (Fig. 1C). The specific activi-
ties of RNase A and DNase were also checked on
plasmid DNA; DNase did indeed have strong activity,
whereas RNase A was not contaminated by DNase
activity (data not shown). We therefore conclude that
the nucleic acid content found in the recombinant
UDE preparation that copurifies with UDE on both
Ni
2+
-agarose and size exclusion chromatography is, in
A
B
CD
Fig. 1. Purification of recombinant UDE and its RNA binding. (A) Elution profile of recombinant UDE in Ni
2+
-affinity chromatography resulted
in two distinct peaks. Fat and thin lines correspond to absorbance of the eluate at 280 and 260 nm, which are characteristic for proteins and
nucleic acids, respectively. The dashed line shows the imidazole gradient applied to promote the elution of His-tagged UDE. Left and right
vertical axes indicate absorbance units and imidazole concentration, respectively. Two peaks eluted at 300 and 600 m
M imidazole, respec-
tively; the first contained significant amount of nucleic acid, and the second was practically free of nucleic acids. The insert shows the pro-
tein contents of the two corresponding fractions (1 and 2) analysed by SDS ⁄ PAGE. (B) Elution profile of the two UDE fractions in size
exclusion chromatography. Fat and thin lines show absorbance of the eluate at 280 nm and 260 nm, respectively. Black and grey lines corre-
spond to the nucleic acid-containing and nucleic acid-free UDE fractions obtained by the previous Ni
2+
-affinity chromatography, respectively.
The observed values of the elution volume characteristic for the two fractions were 9 and 14.5 mL, corresponding to more than 600-kDa
and about 52-kDa apparent molecular masses, respectively. (C) The nucleic acid content of the first fraction of UDE was detected on aga-
rose gel as RNA. Lane 1: nucleic acid containing UDE fraction from Ni
2+
-affinity chromatography. Lane 2: the same treated with DNase.
Lane 3: the same treated with RNase. Lane 4: the same treated with proteinase K. Marker positions are indicated on the right. (D) DNase
and RNase treatment of the nucleic acid containing UDE fraction analysed by size exclusion chromatography also identified the nucleic acid
as RNA. Fat and thin lines show absorbance of the eluate at 280 and 260 nm, respectively. Nucleic acid-containing UDE without treatment
(black), upon DNase treatment (grey) and upon RNase A treatment (light grey) were analysed by size exclusion chromatography. Note that
RNase A may degrade the nucleic acid content of UDE, resulting in a similar elution profile to that of the nucleic acid-free UDE (B). The large
peak at about 20-mL elution volume (light grey chromatogram) might be caused by RNA fragments or nucleotides.
A. Bekesi et al. RNA-dependent conformational states of UDE
FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS 297
fact, RNA. Therefore, we termed the RNA-containing
UDE fraction RNA–UDE and, in parallel, the RNA-
free UDE fraction as UDE.
RNA–UDE was also analysed by size exclusion
chromatography after DNase or RNase treatment
(Fig. 1D). DNase treatment did not cause significant
changes in the chromatogram; one single peak in the
exclusion volume with significant nucleic acid content
(compare absorbance values at 260 and 280 nm) was
detected, similarly to what was seen in the chromato-
gram of the untreated sample. In contrast, RNase
treatment resulted in a drastic decrease in this peak,
with the concomitant emergence of a small addi-
tional peak at the same position as in the case of
untreated RNA-free UDE. The straightforward sepa-
ration of these two UDE species by chromatography
suggested that these species may represent two confor-
mational states.
RNA content of RNA–UDE
On native agarose gel, the RNA content of RNA–
UDE appears as a smear at above 10 000-nucleotide
apparent size (Fig. 1C). However, upon treatment of
RNA–UDE with proteinase K, a band appeared at a
much lower apparent size; this could correspond to
degradation products of the RNA. The observed sig-
nificant gel shift also confirms the binding between the
protein and the RNA in RNA–UDE.
To analyse the composition of RNA–UDE, we per-
formed deconvolution of its UV–visible absorbance
spectrum, using the separately determined A
260 nm
⁄
A
280 nm
ratios (2 for RNA, and 0.5 for nucleic acid-
free UDE, respectively). On the basis of this analysis,
the ratio is about 17–22 RNA nucleotides per UDE
monomer. This also means that several UDE proteins
may bind to the same RNA molecule, suggesting non-
sequence-specific characteristics of the binding.
UDE does not show RNA-cleaving activity
UDE was described as a uracil-DNA-degrading factor.
It was of interest to investigate whether UDE can also
degrade RNA. The results shown in Fig. 1C reveal
that the RNA content found to be associated with
UDE during extraction and purification is present as
100–500 nucleotides long molecular species, indicating
that UDE may not exhibit RNase-like degrading activ-
ity. To further investigate this, we tested the effect of
both RNA–UDE and UDE on a double-stranded
in vitro synthesized RNA of 471 nucleotides. Figure 2
shows that neither RNA–UDE nor UDE possesses
RNA-degrading activity.
The above results indicated that UDE may exist in
two distinct conformations that may not be in dynamic
equilibrium, as these cannot be converted into each
other under our experimental conditions. In one con-
formational state, UDE is present in a complex with
RNA (RNA–UDE), whereas in the other state, the
protein is practically free of bound nucleic acids
(UDE). To determine the specific characteristics of
these two conformational states, we investigated ther-
mal stability, secondary structure, the presence of
exposed hydrophobic surface patches, and the binding
ability of RNA and DNA oligonucleotides with regard
to both RNA–UDE and UDE.
RNA binding significantly affects thermal
unfolding of UDE
We followed the thermal unfolding of gel-filtrated
UDE and RNA–UDE fractions by tryptophan fluores-
cence (Fig. 3A) and by CD spectroscopy (Fig. 3B). In
the case of tryptophan fluorescence, we monitored the
specific local milieu of the four tryptophans within the
sequence (Trp10, Trp107, Trp259 and Trp299 within
the nonconserved N-terminal region, motifs 1A, 3, and
4, respectively [7,8]), and during the CD measure-
ments, we followed the global change in the ratio of
detected secondary structure elements. Despite this
basic dissimilarity between the two methods, the deter-
mined T
m
values were different by only about 2 °C,
indicating that conformational changes during unfold-
ing can be faithfully monitored by both techniques.
We found that RNA–UDE had a considerably lower
melting temperature than UDE. Interestingly, this
lower thermal stability was coupled to much higher
cooperativity during unfolding, as shown by the steep
slope of the RNA–UDE melting transition (Fig. 3 and
Table 1). These results indicated that, in the absence of
Fig. 2. UDE does not have RNase-like activity. Both RNA–UDE and
UDE were tested for RNase-like activity, using 471-bp dsRNA. Incu-
bation times are indicated at the top, and marker positions on the
right side. The observed shift may be caused by the protein–RNA
complex. Note the absence of significant time-dependent degrada-
tion.
RNA-dependent conformational states of UDE A. Bekesi et al.
298 FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS
the nucleic acid ligand, UDE may lose some of its sec-
ondary and tertiary interactions, whereas in the con-
formational state characteristic of RNA–UDE, the
protein exists in a more ordered and potentially meta-
stable state. The lower melting temperature of RNA–
UDE may reflect dissociation of the RNA followed
immediately by the consequent unfolding of the UDE
protein devoid of RNA. Hence, the lower melting
temperature of RNA–UDE might indicate basic
differences between the UDE and RNA–UDE confor-
mations. Importantly, unfolding of RNA–UDE occurs
according to a simple two-state model, indicating an
intimate interaction between RNA and the protein.
Elevated cooperativity in the RNA–UDE state can be
explained by assuming that maintenance of the 3D
protein structure strongly depends on RNA binding.
Such interactions may originate from translational-
coupled folding of UDE.
For further confirmation of distinct conformations
within the two states, RNA isolated from RNA–UDE
(using Trizol; see Experimental procedures) was added
to UDE. In this sample, the melting curve shows the
same phenomenon as in the case of UDE alone
(Fig. 3A). The fact that the conformation specific to
RNA–UDE could not be restored simply by RNA
readdition under the experimental conditions also
suggests an intrinsic relationship between the RNA
binding and protein folding in RNA–UDE.
Interestingly, treatment of RNA–UDE with RNa-
se A did not result in significant changes of the melting
curve, indicating that the characteristics of the confor-
mational state initially detected on the RNA–UDE
sample are preserved, at least for the duration of the
experiments (Fig. 3B). However, the melting curve of
further purified RNase-treated RNA–UDE was charac-
teristic for UDE (Fig. 3B), indicating that a transition
from the RNA–UDE to the UDE conformational state
is possible, even though it is not an immediate process.
On the basis of these results, we propose a scheme of
possible transitions and relations between the two
conformational states of UDE protein (Fig. 4).
RNA–UDE shows an increased amount of helical
content as compared with UDE
To characterize the conformational differences between
RNA–UDE and UDE suggested by thermal stability
data, several potentially relevant spectroscopic meth-
ods were applied. Tryptophan fluorescence showed
practically the same emission maximum wavelength
(data not shown); however, CD spectroscopy revealed
significant differences. After accurate determination of
protein concentrations from UV spectra as well as the
Bradford assay (which were in agreement within 7%
standard error) and densitometry of SDS ⁄ PAGE
bands, CD spectra were measured in the 190–270-nm
far-UV range (Fig. 5A). Although spectra of both
RNA–UDE and UDE showed a-helical characteristics,
a clear difference was also detected between the two
spectra, reflected by: (a) different signal intensities; and
(b) different shapes, e.g. a slight red shift in the
position of the 208-nm peak (Fig. S1). Quantitative
evaluation of the CD data is shown in the bar graph
A
B
Fig. 3. Effect of RNA binding on the thermostability of UDE.
(A) Thermal denaturation followed by tryptophan fluorescence.
Normalized and corrected curves are shown for RNA–UDE (full
black circles), UDE (full grey squares), UDE + RNA mixture (open
triangles) and RNase-treated RNA–UDE after repurification by size
exclusion chromatography (open squares). (B) Thermal denaturation
followed by CD spectroscopy. Curves are shown for RNA–UDE (full
black circles), UDE (full grey squares), in situ treatment of RNA–
UDE with RNase A (open circles), and RNase-treated RNA–UDE
after repurification by size exclusion chromatography (open
squares). The lines show the results of sigmoidal fitting. Insert: CD
spectra of RNA–UDE (black) and UDE (grey) in the native (20 °C,
full symbols) and denatured (70 °C, open symbols) states; lines
show fitted spectra calculated by
CDSSTR software [9].
A. Bekesi et al. RNA-dependent conformational states of UDE
FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS 299
of Fig. 5A, where the secondary structural elements
termed Helix1, Helix2, Strand1 and Strand2 corre-
spond to the two subcategories of helices and sheets
(regular and distorted fractions) as defined in [9].
RNA–UDE showed approximately 20% higher a-heli-
cal content than UDE. As accurate measurement of
protein concentration, especially when the protein is in
complex with nucleic acids, is not trivial, the suggested
conformational changes between RNA–UDE and
UDE are much strengthened in the case of significantly
different concentration-independent intensive parame-
ters. Such differences are shown in Fig. S1 (shape of
the far-UV CD spectra) and in Fig. 3 (T
m
and cooper-
ativity of thermal unfolding).
To test the model represented in Fig. 4, the respec-
tive samples were produced and characterized by CD.
A UDE+RNA mixture failed to restore the CD signal
associated with RNA–UDE (Fig. 5A, top left). RNA
alone did not result in significant CD spectra, as
shown in Fig. S1. In situ RNase treatment of RNA–
UDE resulted in an intermediate spectrum (Fig. 5A,
top right), whereas after gel filtration, this sample pro-
vided practically the same spectrum as observed for
UDE (Fig. 5A, bottom left). The same findings were
obtained by quantitative evaluation (Fig. 5A, bar
graph, bottom right). These results are in agreement
with the thermal unfolding studies (Fig. 3) and rein-
force the model of the two conformational states.
The presence of hydrophobic surface cavities is
significantly increased in RNA–UDE as compared
with UDE
To determine whether the above described differences
in the conformational states of UDE are also reflected
on the protein surface, we evaluated the interactions of
UDE and RNA–UDE with the environmentally sensi-
tive protein dyes 8-anilinonaphthalene-1-sulfonate
(ANS) and Sypro Orange. ANS is known to bind to
NH
3
+
moieties of the proteins, and exhibits elevated
fluorescence if this binding occurs in a hydrophobic
microenvironment [10]. Sypro Orange is known as a
protein gel stain (it does not bind to either nucleic
acids or lipids [11]), but it can be used as an alternative
to ANS in the analysis of hydrophobic protein surfaces
Table 1. Melting temperatures and values of cooperativity characteristics for thermal transitions of different conformational states of UDE
protein. The value for cooperativity come from the dT parameter of the sigmoidal fit of the melting curve, which negatively correlates with
the cooperativity of the thermal transition. Error values were derived from the average of several independent measurements from different
UDE protein preparations. ND, non determined.
RNA–UDE UDE
UDE + isolated
RNA
In situ RNase-treated
RNA–UDE
RNase-treated
RNA–UDE, purified
Melting temperature (°C)
CD 50.2 ± 1.5 55.2 ± 0.6 ND 48.3 ± 0.5 55.3 ± 1.3
Fluorimetry 51.5 ± 0.6 56.6 ± 0.5 57.05 ± 0.13 ND 55.5 ± 0.3
Relative cooperativity (1 ⁄ dT )
CD 0.58 ± 0.03 0.21 ± 0.03 ND 0.45 ± 0.06 0.22 ± 0.04
Fluorimetry 0.60 ± 0.05 0.34 ± 0.02 0.27 ± 0.01 ND 0.36 ± 0.01
Fig. 4. Scheme of possible transitions between the two conforma-
tional states. The conformational state of RNA–UDE (black moon-like
shape, top left) is not destroyed immediately upon RNase treatment
(bottom left). After removal of RNA fragments (grey curves, bottom
left), the protein conformation changes into one characteristic for
UDE (grey circular segment, bottom right). UDE can bind to RNA
(grey curve, top right), but cannot be transformed into the specific
RNA UDE complex present in the RNA-UDE state.
RNA-dependent conformational states of UDE A. Bekesi et al.
300 FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS
[12]. Addition of either ANS or Sypro Orange to
RNA–UDE led to a drastic increase in the fluorescence
emission of the dyes, whereas a much smaller incre-
ment was induced upon mixing of the dyes with UDE
(Fig. 5B). A clear difference was also observed in the
positions of emission maxima characteristic for
A
B
Fig. 5. Conformational differences between RNA-UDE and UDE. (A) Secondary structure elements characteristic for RNA-UDE and UDE calcu-
lated from far-UV CD spectra. Top left: spectra for RNA-UDE (full black circles), UDE (full grey squares), and UDE + RNA mixture (open trian-
gles). Top right: spectra for RNA-UDE (as above), UDE (as above), and RNase-treated RNA-UDE (open black circles). Bottom left: spectra for
RNA-UDE (as above), UDE (as above), and RNase-treated and repurified RNA-UDE (open grey circles). Lines on the spectra indicate fitted
curves calculated by
CDSSTR software at the DICHROWEB server. The ratio of secondary structure elements, indicated on the bar graph (bottom
right), was calculated from several independent spectra in each case. The terms Helix1, Helix2, Strand1 and Strand2 correspond to the two
subcategories of helices and sheets (regular and distorted fractions) as defined in [9,71]. On the bar graph, RNA-UDE (black), UDE (grey),
UDE + RNA mixture (hatched grey), RNase-treated RNA-UDE (hatched black) and RNase-treated and repurified RNA-UDE (light grey) are
shown. (B) Altered hydrophobic surface patches in RNA-UDE and UDE. Left panel: spectra of 1.5 l
M RNA-UDE (black curves) and 1.5 lM UDE
(grey curves) mixed with 200 l
M ANS. Spectral maxima were at 471 ± 2 and 482 ± 1 nm for RNA-UDE and UDE, respectively. Right panel:
ANS titration. RNA-UDE (full black circles), UDE (full grey squares), RNase-treated (open circles), and UDE + RNA mixture (open triangles).
Maximal fluorescent signal intensities of the individual spectra are shown for each titration point. Lines show hyperbolic fitting of the data.
A. Bekesi et al. RNA-dependent conformational states of UDE
FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS 301
RNA–UDE and UDE complexed with both dyes [for
ANS, 471 and 482 nm, respectively (Fig. 5B); and
for Sypro Orange, 567 and 583 nm, respectively
(Fig. S2A)]. These results indicated that the number of
hydrophobic surface cavities may be much increased in
RNA–UDE as compared with UDE. We can exclude
the possibility that putative dimerization of UDE may
hide the hydrophobic surface exposed in the RNA–
UDE state, as full-length UDE was shown to be in a
monomeric state in solution [8].
For a detailed analysis, titration experiments were
performed with both dyes (Figs 5B and S1B). The
maximum emission values were plotted against dye
concentration, and the results were fitted with hyper-
bola (Figs 5B and S1B; Table 2).
The apparent dissociation constant for ANS and
RNA–UDE was three-fold smaller than that for UDE,
and the emission maximum was red-shifted from 468
to 478 nm, whereas, in the case of UDE, the emission
maximum was not shifted, also indicating altered ANS
binding fashion of the two conformational states of
UDE protein. Upon treatment of RNA–UDE with
RNase, only a slight decrease was detected in the fluo-
rescent signals (Fig. 5B), indicating that the conforma-
tional state characteristic for RNA–UDE was not
completely disrupted. When isolated RNA, in an
amount equivalent to that in RNA–UDE, was added
to UDE, we observed a significant increase in fluores-
cence; however, the extent of the signal was about half
that seen with the RNA–UDE sample, and the appar-
ent dissociation constant was more than two-fold
higher (Fig. 5B and Table 2).
Although titration with Sypro Orange resulted in
better-quality signals, evaluation of these curves can
only provide relative data, because the dye concentra-
tion data is not given. Sypro Orange complexed with
RNA–UDE showed a similar apparent dissociation
constant, but a much higher fluorescent signal inten-
sity, more than 20-fold higher than that with the Sypro
Orange–UDE complex (Fig. S2B; Table 2). The emis-
sion maximum was red-shifted from 567 to 587 nm,
whereas, in the case of UDE, the emission maximum
was not shifted. These results also support the altered
surface hydrophobicity in the two conformational
states.
Upon treatment of RNA–UDE with RNase, very
similar large fluorescent signals were detected, indicat-
ing that the conformational state characteristic for
RNA–UDE was not disrupted in the RNase-treated
mixture (Fig. S2B). This observation is in agreement
with the results of ANS titration measurements
(Fig. 5B), the melting experiments (Fig. 3), and CD
spectra (Fig. 5A).
When isolated RNA was added to UDE, we
observed a significant increase in fluorescence; how-
ever, the extent of the signal was much smaller than
that with the RNA–UDE sample, and the apparent
dissociation constant was seven-fold higher (Table 2;
Fig. S2B). This finding also suggests that the confor-
mational state of RNA–UDE, which showed a high
fluorescence intensity in this assay, could not be recon-
structed by simple addition of the isolated RNA com-
ponent to UDE, potentially indicating that the
conformation of RNA–UDE may originate in de novo
protein folding during translation.
In summary, the results obtained in complexation
experiments with ANS and Sypro Orange are in excel-
lent agreement with the hypothesis of the two confor-
mational states presented in Fig. 4, and are also in line
with the findings of the thermal unfolding and CD
studies.
RNA binding causes similar protection against
limited proteolysis as DNA binding
Previously, we performed limited proteolysis of UDE
and its complex with DNA, using AspN endoprotein-
ase and high-specificity chymotrypsin, and revealed the
contribution of N-terminal motifs 1A and 1B to DNA
binding as well as the relative compactness of the
C-terminal segment containing motifs 2, 3, and 4 [8].
Here, we aimed at characterization of proteolytic frag-
ments independently in the RNA–UDE and UDE
fractions, using AspN and ArgC endoproteinases
(Fig. 6). In UDE, several preferred proteolytic cleavage
sites were identified by MS (summarized in Fig. 6C),
whereas in RNA–UDE, protection against both pro-
teinases was evident on motifs 1A and 1B. The rela-
tive compactness of the C-terminal part containing
motifs 2, 3 and 4 was confirmed in both RNA–UDE
Table 2. Hydrophobic surface titration of different conformational
states of UDE protein, using ANS and Sypro Orange dyes. Parame-
ters were obtained by hyperbolic fitting of the data. F
max
values indi-
cate the maximal fold increase in fluorescence intensity. K
app
values
are apparent dissociation constants for dye–protein complexes.
Protein
Parameters
ANS Sypro Orange
F
max
(· 1000) K
app
F
max
(· 1000) K
app
UDE 18.7 ± 0.3 208 ± 5 3.8 ± 0.2 34 ± 7
RNase-treated
RNA–UDE
31.5 ± 1.0 62 ± 10 71.6 ± 3.3 44 ± 5
RNA–UDE 42.5 ± 0.5 77 ± 2 68.3 ± 4.2 47 ± 7
UDE + RNA 27.6 ± 0.9 161 ± 9 46.6 ± 7.2 300 ± 75
RNA-dependent conformational states of UDE A. Bekesi et al.
302 FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS
and UDE, as preferred cleavage sites could not be
identified in this region. To characterize the relation-
ship between the RNA-binding and DNA-binding
sites, DNA was added to both RNA–UDE and UDE.
The proteolysis patterns were rather similar in both
RNA-containing and DNA-containing complexes, with
one remarkable difference in AspN digestion. Here,
RNA binding produced significant protection at the
Asp333 site within the nonconserved C-terminus,
whereas DNA binding did not induce any protection
(Fig. 6B). These results suggest partially, but not fully,
overlapping sites for RNA and DNA binding in UDE
protein. Furthermore, addition of DNA in three-fold
molar excess to RNA–UDE results in exactly the same
patterns as those characteristic for the UDE–DNA
complex (Fig. 6A,B), suggesting that DNA is able to
replace RNA in RNA–UDE, in agreement with the
overlap of the respective binding sites. These findings
can be interpreted within the previously introduced
hypothesis that RNA–UDE and UDE represent two
distinct conformational states, although they do not
offer independent support in this respect.
Characterization of the nucleic acid-binding
ability of UDE
In order to provide a quantitative description of UDE
DNA-binding and RNA-binding properties, we
applied EMSA. In the first set of experiments, we
assessed the interaction of UDE and RNA–UDE with
DNA oligonucleotides (Fig. 7). When a double-
stranded 30mer oligonucleotide (see Experimental pro-
cedures) was titrated with UDE, we detected two
shifted bands (termed complex 1 and complex 2,
respectively) on 6% native TBE ⁄ PAGE (Fig. 7A).
Densitometry of the bands corresponding to the free
and complexed oligonucleotides resulted in a sigmoidal
decrease for the amount of free oligonucleotide, and a
A
B
C
Fig. 6. Comparison of RNA-binding and DNA-binding surfaces by limited proteolysis. (A, B) Limited proteolysis using ArgC and AspN endo-
proteinases. Molecular mass marker positions in kDa are indicated on the left. U-pl, uracil-substituted plasmid DNA mixed with the protein
samples prior to digestion. The first lane of each gel represents untreated samples; further lanes correspond to 10-min, 1-h, 3-h and 5-h time
points. The black cross and the black star indicate RNA protection against first cleavage at the C-terminus by ArgC and AspN, respectively.
Note that the DNA binding does not protect these sites. (C) Scheme of preferred cleavage sites. Cleavage sites were identified by MS. The
scheme was designed to be strictly proportional to the real sizes. His: His-tag. 1A, 1B, 2, 3, and 4: conserved motifs of UDE. NLS: nuclear
localization signal at the end of the sequence [17]. Black and grey arrows indicate the preferred cleavage sites of ArgC and AspN, respec-
tively. Solid arrows: preferred sites even in the presence of nucleic acids. Dotted arrows: sites protected by both RNA and DNA binding.
Dashed arrows: sites protected by RNA binding, but not by DNA binding. Numbering of residues follows the sequence of the physiological
form derived from the NCBI database. R* indicates the arginine present only in the His-tagged protein. In the absence of straightforward
identification of close cleavage sites, arrows list multiple potential sites.
A. Bekesi et al. RNA-dependent conformational states of UDE
FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS 303
sigmoidal increase for complex 2, whereas complex 1
was only transiently present, in a manner characteristic
for intermediate states (Fig. 7B). Data were fitted with
the Hill equation (Fig. 7C; see Experimental proce-
dures), providing an apparent K
d
value of
50±13lm, and n = 4.9 ± 0.2. The value of the
apparent Hill coefficient may indicate either complexes
with higher stoichiometry or strong cooperativity [13]
(see Experimental procedures). Figure 7A shows only
two bands corresponding to complex forms; however,
the upper band, located in the well without significant
migration, may contain additional complex forms pro-
posed by the apparent Hill coefficient. To further
address the stoichiometry issue, a similar EMSA was
A
B
C
D
E
Fig. 7. DNA-binding ability of UDE and RNA–UDE characterized by EMSA. (A) UDE causes a significant shift in the electrophoretic mobility
of a 30mer oligonucleotide. One-micromolar 30mer double-stranded oligonucleotide with a single uracil at the mid-position was titrated with
increasing amounts of UDE, up to 5 l
M, in native 6% TBE ⁄ PAGE. Arrows on the left show the positions of free oligonucleotide and two dis-
tinct complex forms. (B) Densitometry of the bands corresponding to the three detected species. Band densities (free oligonucleotide, full
black squares; complex 1, grey circles; complex 2, open squares) were normalized and plotted against UDE concentration. (C) Evaluation of
binding. The total relative amount of bound oligonucleotide (black squares) was calculated from the Eqn (1) – [free], where [free] is the rela-
tive amount of free oligonucleotide, and plotted against UDE concentration. The line shows the curve fitted by the Hill equation. (D) EMSA
on agarose gel revealed the presence of several complex forms with higher stoichiometry. Two-micromolar 30mer (left panel) and 60mer
(right panel) oligonucleotides with a single uracil at the mid-position were compared on 1.5% agarose gel, with UDE concentrations up to
32 l
M. Symbols (rhomboids) at both sides indicate positions of complexes; five and seven distinct positions were observed for 30mer and
60mer oligonucleotides, respectively. (E) DNA-binding ability of UDE versus RNA–UDE. One-micromolar Cy3-labelled single-stranded uracil-
containing oligonucleotide was titrated with UDE (top panel) and RNA–UDE (bottom panel) with protein concentrations up to 5.5 l
M. Note
that, in the case of RNA–UDE, saturation was not observed.
RNA-dependent conformational states of UDE A. Bekesi et al.
304 FEBS Journal 278 (2011) 295–315 ª 2010 The Authors Journal compilation ª 2010 FEBS
[...]... of mRNA in protein folding On the basis of the above detailed results and data from the literature, we conclude that the RNAUDE complex may be formed via a cofolding mechanism The present results shed light on novel aspects of UDE structure, providing important insights into its RNAdependent native fold; the protein is also identied as a model for future studies of RNA- assisted protein folding Furthermore,... de novo protein folding during translation To characterize the RNA species copuried with the protein, the presence of three specic RNA molecules was checked by reverse transcription followed by realtime PCR We targeted the mRNA of UDE, which is abundant in this recombinant overexpression system; domain V of the 23S rRNA, which is known to be involved in assisting de novo protein folding during translation... on the surface of the ribosome as the polypeptide chain reaches the exit site [4850] Within the ribosomal tunnel, 23S rRNA interacts with the nascent polypeptide chain, helping secondary structure formation Moreover, direct interaction of rRNA with proteins, especially with nucleic acid-binding proteins, also has an effect on protein refolding in vitro [51,52] On the ribosomal surface, large-scale folding. .. characterization of UDE localization in tissues awaits further studies Here, we addressed the possible impact of RNA binding on protein structure and substrate-binding ability Proteins that bind and process nucleic acids are often capable of binding to both RNA and singlestranded or double-stranded DNA, producing altered biological functions, depending on the type of ligand Several proteins are known... the folding of exible or disordered regions, especially in the case of nucleic acid-binding proteins (e.g bateriophage lambda integrase [43], GCN4-bZIP transcription factor [44,45], and RNase P [46]) Here, we found that RNA binding alters the protein conformation, signicantly enhancing its a-helical content (Fig 5), strengthens the cooperativity of protein thermal unfolding, and lowers the melting... threshold of 2000 are 20.4, 21.5 and 32.2 for the three targets, respectively, indicating that rRNA may be present in more than two-fold excess over mRNA of UDE, and that the presence of mRNA of GAPDH is negligible (< 0.5% as compared with rRNA) For analysis of the composition of the bound RNA pool with an independent method, the isolated RNA was converted into double-stranded cDNA, using both random hexamer... relevance of UDE RNA binding Fig 9 Determination of the RNA content of RNAUDE by realtime RT-PCR The RNA content of RNAUDE was isolated and reverse-transcribed, using a random hexamer primer Real-time PCR amplication curves are shown, obtained with primers specic for 200-nucleotide segments of either domain V of 23S rRNA (grey line), mRNA of UDE (black line), or mRNA of GAPDH (light grey line) Ct values... demonstrated in both the heterologous overexpression system in E coli and in fruit y cellular extracts (Fig 10) In the E coli recombinant system, the bound RNA pool was shown to consist of 16S rRNA, 23S rRNA, and UDE mRNA (Fig 9; Table 3), potentially implicating either an additional RNA- related function or RNA- binding-coupled folding of UDE protein Indepth analysis revealed signicant conformational. .. [21], H-NS [22], and Y-box binding proteins [23,24]) are also involved in post-transcriptional regulation, owing to their alternative RNA- binding ability Human AP endonuclease I, also referred to as Ref1 transcription factor, also binds to and cleaves RNA [25] Importantly, the physiological functions of several proteins directly involve both DNA-binding and RNA- binding ability (telomerase, RNA- dependent... between RNAUDE and UDE, the two distinct fractions separable by independent chromatography methods Furthermore, we demonstrated that the specic conformation characteristic for RNAUDE cannot be generated simply by addition of the RNA to UDE (Figs 3, 5, and S1), which also argued for the possibility of RNA- assisted cotranslational folding Ligand binding often stabilizes the protein structures by enhancing the . Association of RNA with the uracil-DNA-degrading factor
has major conformational effects and is potentially
involved in protein folding
Angela. an intrinsic relationship between the RNA
binding and protein folding in RNA UDE.
Interestingly, treatment of RNA UDE with RNa-
se A did not result in
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