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
Trang 1has major conformational effects and is potentially
involved in protein folding
Angela Bekesi1, Maria Pukancsik1, Peter Haasz1, Lilla Felfoldi1, Ibolya Leveles1, Villo Muha1,
Eva Hunyadi-Gulyas2, Anna Erdei3, Katalin F Medzihradszky2,4and Beata G Vertessy1,5
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
identi-of a new protein family with unique enzyme activity that has a putativerole in insect development In addition, UDE may also serve as potentialtool in molecular biological applications Owing to lack of homology withother proteins with known structure and⁄ or function, de novo data arerequired 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 ofRNA strongly bound to the protein, influencing its conformation Detailedbiophysical 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, implyingputatively 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 thetwo 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 successfulcrystallization; on the other hand, it might imply a further molecular func-tion of the protein In fact, RNA-dependent complexation of UDE wasalso 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.
Trang 2a 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, asshown by thermal transitions monitored by CD andfluorescence spectroscopy Possible effects of RNAbinding on UDE conformation were assayed by CDspectroscopy and by titration of hydrophobic surfacepatches The DNA-binding and RNA-binding abilities
of the enzyme forms were measured by electrophoreticmobility shift assay (EMSA) Characterization of thebound RNA pool by real-time RT-PCR revealed anabundance of UDE mRNA as well as of domain V of23S rRNA These results, together with the character-istics of the two distinct conformational states, suggestputative cofolding of UDE and RNA during transla-tion Physiological relevance of RNA–UDE bindingwas also demonstrated
Results
Two distinct molecular populations of UDE can
be separated by Ni2+-affinity and size exclusionchromatography
In a refined purification method with recombinant tagged UDE (see Experimental procedures), gradientelution was applied on a HisTrap column with AKTApurifier Two distinct fractions of intact full-lengthUDE eluted at about 300 and 600 mm imidazole,respectively (Fig 1A), reflecting their altered affinityfor the Ni2+-column SDS⁄ PAGE indicated the sameelectrophoretic mobility for UDE in both fractions,arguing against protein degradation or observablemodifications (Fig 1A, insert panel) MS analysis ofthe two protein bands further confirmed the lack ofany detectable post-translational modifications (datanot shown) In the first fraction, UDE eluted togetherwith a large amount of nucleic acid, whereas the sec-ond fraction was practically nucleic acid-free Bothfractions were subjected to size exclusion chromatogra-phy, where the nucleic acid-containing fraction eluted
His-in the exclusion volume (correspondHis-ing to an apparentmolecular mass larger than 600 kDa), and the nucleicacid-free fraction eluted at a position corresponding to
an apparent molecular mass of 52 kDa (Fig 1B) viously, we reported purification of UDE by stepwise
Pre-Ni2+-agarose chromatography, which did not allowseparation of these two fractions [8] Size exclusionchromatography of this UDE preparation resulted intwo distinct peaks at the same positions as above Theobservation that full-length UDE can be separated intotwo fractions by applying two different chromato-graphic methods, Ni2+-agarose affinity and size
Trang 3exclusion, 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 aswell as by RNase A were performed Surprisingly,DNase treatment did not significantly perturb thedetected nucleic acid band, whereas RNase treatmentcompletely eliminated it (Fig 1C) The specific activi-ties of RNase A and DNase were also checked onplasmid DNA; DNase did indeed have strong activity,whereas RNase A was not contaminated by DNaseactivity (data not shown) We therefore conclude thatthe nucleic acid content found in the recombinantUDE preparation that copurifies with UDE on both
Ni2+-agarose and size exclusion chromatography is, in
Fig 1 Purification of recombinant UDE and its RNA binding (A) Elution profile of recombinant UDE in Ni2+-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 Ni2+-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.
Trang 4fact, 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 A260 nm⁄
A280 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 intwo distinct conformations that may not be in dynamicequilibrium, as these cannot be converted into eachother under our experimental conditions In one con-formational state, UDE is present in a complex withRNA (RNA–UDE), whereas in the other state, theprotein is practically free of bound nucleic acids(UDE) To determine the specific characteristics ofthese two conformational states, we investigated ther-mal stability, secondary structure, the presence ofexposed hydrophobic surface patches, and the bindingability of RNA and DNA oligonucleotides with regard
to both RNA–UDE and UDE
RNA binding significantly affects thermalunfolding of UDE
We followed the thermal unfolding of gel-filtratedUDE and RNA–UDE fractions by tryptophan fluores-cence (Fig 3A) and by CD spectroscopy (Fig 3B) Inthe case of tryptophan fluorescence, we monitored thespecific local milieu of the four tryptophans within thesequence (Trp10, Trp107, Trp259 and Trp299 withinthe nonconserved N-terminal region, motifs 1A, 3, and
4, respectively [7,8]), and during the CD ments, we followed the global change in the ratio ofdetected secondary structure elements Despite thisbasic dissimilarity between the two methods, the deter-mined Tm values were different by only about 2C,indicating that conformational changes during unfold-ing can be faithfully monitored by both techniques
measure-We found that RNA–UDE had a considerably lowermelting temperature than UDE Interestingly, thislower thermal stability was coupled to much highercooperativity during unfolding, as shown by the steepslope of the RNA–UDE melting transition (Fig 3 andTable 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.
Trang 5the 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 occursaccording to a simple two-state model, indicating anintimate interaction between RNA and the protein.Elevated cooperativity in the RNA–UDE state can beexplained by assuming that maintenance of the 3Dprotein structure strongly depends on RNA binding.Such interactions may originate from translational-coupled folding of UDE
For further confirmation of distinct conformationswithin the two states, RNA isolated from RNA–UDE(using Trizol; see Experimental procedures) was added
to UDE In this sample, the melting curve shows thesame phenomenon as in the case of UDE alone(Fig 3A) The fact that the conformation specific toRNA–UDE could not be restored simply by RNAreaddition under the experimental conditions alsosuggests an intrinsic relationship between the RNAbinding and protein folding in RNA–UDE
Interestingly, treatment of RNA–UDE with
RNa-se A did not result in significant changes of the meltingcurve, indicating that the characteristics of the confor-mational state initially detected on the RNA–UDEsample are preserved, at least for the duration of theexperiments (Fig 3B) However, the melting curve offurther purified RNase-treated RNA–UDE was charac-teristic for UDE (Fig 3B), indicating that a transitionfrom 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 ofpossible transitions and relations between the twoconformational states of UDE protein (Fig 4)
RNA–UDE shows an increased amount of helicalcontent as compared with UDE
To characterize the conformational differences betweenRNA–UDE and UDE suggested by thermal stabilitydata, several potentially relevant spectroscopic meth-ods were applied Tryptophan fluorescence showedpractically the same emission maximum wavelength(data not shown); however, CD spectroscopy revealedsignificant differences After accurate determination ofprotein concentrations from UV spectra as well as theBradford assay (which were in agreement within 7%standard error) and densitometry of SDS⁄ PAGEbands, CD spectra were measured in the 190–270-nmfar-UV range (Fig 5A) Although spectra of bothRNA–UDE and UDE showed a-helical characteristics,
a clear difference was also detected between the twospectra, reflected by: (a) different signal intensities; and(b) different shapes, e.g a slight red shift in theposition of the 208-nm peak (Fig S1) Quantitativeevaluation 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].
Trang 6of 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 incomplex with nucleic acids, is not trivial, the suggestedconformational changes between RNA–UDE andUDE are much strengthened in the case of significantlydifferent concentration-independent intensive parame-ters Such differences are shown in Fig S1 (shape ofthe far-UV CD spectra) and in Fig 3 (Tmand cooper-ativity of thermal unfolding)
To test the model represented in Fig 4, the tive samples were produced and characterized by CD
respec-A UDE+RNrespec-A mixture failed to restore the CD signalassociated with RNA–UDE (Fig 5A, top left) RNAalone did not result in significant CD spectra, asshown 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 forUDE (Fig 5A, bottom left) The same findings wereobtained by quantitative evaluation (Fig 5A, bargraph, bottom right) These results are in agreementwith the thermal unfolding studies (Fig 3) and rein-force the model of the two conformational states
The presence of hydrophobic surface cavities issignificantly increased in RNA–UDE as comparedwith 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 ofUDE and RNA–UDE with the environmentally sensi-tive protein dyes 8-anilinonaphthalene-1-sulfonate(ANS) and Sypro Orange ANS is known to bind to
NH3+ moieties of the proteins, and exhibits elevatedfluorescence if this binding occurs in a hydrophobicmicroenvironment [10] Sypro Orange is known as aprotein gel stain (it does not bind to either nucleicacids 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.
UDE + isolated RNA
In situ RNase-treated RNA–UDE
RNase-treated RNA–UDE, purified Melting temperature (C)
Fig 4 Scheme of possible transitions between the two
conforma-tional states The conformaconforma-tional 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.
Trang 7[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 thepositions 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 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 l M 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.
Trang 8calcu-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 sity, more than 20-fold higher than that with the SyproOrange–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 maximumwas not shifted These results also support the alteredsurface hydrophobicity in the two conformationalstates
inten-Upon treatment of RNA–UDE with RNase, verysimilar large fluorescent signals were detected, indicat-ing that the conformational state characteristic forRNA–UDE was not disrupted in the RNase-treatedmixture (Fig S2B) This observation is in agreementwith the results of ANS titration measurements(Fig 5B), the melting experiments (Fig 3), and CDspectra (Fig 5A)
When isolated RNA was added to UDE, weobserved a significant increase in fluorescence; how-ever, the extent of the signal was much smaller thanthat with the RNA–UDE sample, and the apparentdissociation constant was seven-fold higher (Table 2;Fig S2B) This finding also suggests that the confor-mational state of RNA–UDE, which showed a highfluorescence intensity in this assay, could not be recon-structed by simple addition of the isolated RNA com-ponent to UDE, potentially indicating that theconformation of RNA–UDE may originate in de novoprotein folding during translation
In summary, the results obtained in complexationexperiments 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 linewith the findings of the thermal unfolding and CDstudies
RNA binding causes similar protection againstlimited proteolysis as DNA binding
Previously, we performed limited proteolysis of UDEand its complex with DNA, using AspN endoprotein-ase and high-specificity chymotrypsin, and revealed thecontribution of N-terminal motifs 1A and 1B to DNAbinding as well as the relative compactness of theC-terminal segment containing motifs 2, 3, and 4 [8].Here, we aimed at characterization of proteolytic frag-ments independently in the RNA–UDE and UDEfractions, using AspN and ArgC endoproteinases(Fig 6) In UDE, several preferred proteolytic cleavagesites 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 containingmotifs 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 Fmaxvalues
indi-cate the maximal fold increase in fluorescence intensity Kappvalues
are apparent dissociation constants for dye–protein complexes.
Protein
Parameters
Fmax(· 1000) Kapp Fmax(· 1000) Kapp
Trang 9and 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 twodistinct conformational states, although they do notoffer independent support in this respect
Characterization of the nucleic acid-bindingability of UDE
In order to provide a quantitative description of UDEDNA-binding and RNA-binding properties, weapplied EMSA In the first set of experiments, weassessed the interaction of UDE and RNA–UDE withDNA oligonucleotides (Fig 7) When a double-stranded 30mer oligonucleotide (see Experimental pro-cedures) was titrated with UDE, we detected twoshifted bands (termed complex 1 and complex 2,respectively) on 6% native TBE⁄ PAGE (Fig 7A).Densitometry of the bands corresponding to the freeand complexed oligonucleotides resulted in a sigmoidaldecrease for the amount of free oligonucleotide, and a
Trang 10endo-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 Kd value of
50 ± 13 lm, 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 onlytwo bands corresponding to complex forms; however,the upper band, located in the well without significantmigration, may contain additional complex forms pro-posed by the apparent Hill coefficient To furtheraddress the stoichiometry issue, a similar EMSA was
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.