Báo cáo khoa học: Association of RNA with the uracil-DNA-degrading factor has major conformational effects and is potentially involved in protein folding pot

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