Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro Guadalupe de Dios-Bravo 1,2 , Juan Pedro Luna-Arias 3 , Ana Marı ´a Rivero ´ n 4 , Jose ´ J Olivares-Trejo 5 , Ce ´ sar Lo ´ pez-Camarillo 2 and Esther Orozco 5 1 Programa de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatı ´ a del Instituto Polite ´ cnico Nacional, Me ´ xico 2 Programa de Ciencias Geno ´ micas, Universidad de la Ciudad de Me ´ xico, Me ´ xico 3 Departamento de Biologı ´ a Celular, Centro de Investigacio ´ n y de Estudios Avanzados, Me ´ xico 4 Departamento de Biologı ´ a Molecular, Centro Nacional de Investigacio ´ n Cientı ´ fica (CNIC), Habana, Cuba 5 Departamento de Patologia Experimental, Centro de Investigacio ´ n y de Estudios Avanzados, Me ´ xico Entamoeba histolytica is the protozoan responsible for human amoebiasis. E. histolytica strains have distinct capacity to damage cultured cells and human tissues [1–4]. Expression of many molecules and cellular func- tions involved in E. histolytica pathogenicity such as lectins [5,6], adherence molecules [7], proteases [8,9] and amoebapores [10] correlates with its virulence. Variability in virulence exhibited by E. histolytica strains might be controlled in part by transcription of these and other virulence genes. Transcription factors cooperate with other proteins to regulate gene expression. First, the preinitiation complex (PIC) is positioned around the transcription initiation site and then, PIC interacts with other proteins bound to upstream motifs to facilitate the RNA polymerase II function. The absence or the pres- ence of some nuclear factors interacting with PIC may inhibit or promote gene expression to modulate cellu- lar functions [11–13]. Mechanisms, molecules and DNA sequences controlling the spatial and temporal Keywords Entamoeba histolytica; K D ; promiscuous DNA-binding activity; TATA-binding protein; TATA variants Correspondence Esther Orozco, Departamento de Patologı ´ a Experimental, Centro de Investigacio ´ nyde Estudios Avanzados, IPN. C. P. 07360, Me ´ xico, D. F. Fax: +52 55 57477108 Tel: +52 55 50613800 ext 5642 E-mail: esther@mail.cinvestav.mx (Received 23 June 2004, revised 8 December 2004, accepted 11 January 2005) doi:10.1111/j.1742-4658.2005.04566.x The ability of Entamoeba histolytica TATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in super- shift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dis- sociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (±0.39) · 10 )11 and 1.60 (±0.37) · 10 )10 m. TATTTAAA and TAT_ _AAA motifs presented the lowest K D values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding. Abbreviations EhTBP, Entamoeba histolytica TATA-box binding protein; EMSA, electrophoretic mobility shift assays; rEhTBP, recombinant Entamoeba histolytica TATA-box binding protein; NE, nuclear extracts. 1354 FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS transcription patterns during growth, differentiation and development have been widely studied [14,15]. In eukaryotes, general transcription factors such as TFIID ⁄ TFIIB, TFIIA, TFIIE, TFIIF ⁄ RNA poly- merase II and TFIIH are assembled on the core pro- moter before transcription begins [16,17]. The TATA binding protein (TBP) is the first fac- tor that binds DNA to recruit proteins on PIC and initiate gene transcription [18,19]. Mammalian TBPs can productively bind to a large number of diverse TATA elements. An exhaustive statistical genomic survey documented that the TATA box is an A ⁄ T- rich 8 bp segment, often flanked by G⁄ C-rich sequences [20]. Certain E. histolytica genes are activated or down regulated during liver abscesses production by tro- phozoites [9] and during epithelia colonization and invasion. However, we ignore which transcription factors modulate these events and others related to the parasite survival such as trophozoites differenti- ation into cysts. Few transcription factors have been detected and cloned in E. histolytica. URE3-BP, EhEBP1 and EhEBP2 proteins regulate the hgl5 gene expression [21,22] and an EhC ⁄ EBP-like protein is involved in EhPgp1 gene activation [23,24]. Addition- ally, Ehtbp [25] and Ehp53 [26] have been character- ized as the orthologous of the mammalian tbp and p53 genes, respectively. The E. histolytica TATA- binding protein (EhTBP) is the only member of the basal transcription machinery cloned and character- ized in this parasite. The EhTBP functional DNA-binding domain has 55% homology with human TBP, whereas the EhTBP N-terminal domain is rich in hydrophobic residues and quite different from mammalian TBPs [25]. EhTBP C-terminus displays a predicted protein structure fitting to the crystallized human TBP [27,28], but its biological activity has been poorly studied. E. histolytica gene promoters have the TATTTAAA sequence, which is considered as the canonical TATA box for EhTBP [29]. However, EhTBP binding to this sequence has not been fully demonstrated. In addition, TATTTAAA variants have been found surrounding the core promoter, suggesting that these motifs could also act as TATA boxes [30,31], but EhTBP affinity for these sequences is also unknown. In this paper, we studied the in vitro EhTBP binding affinity for differ- ent TATA sequences found in E. histolytica gene pro- moters and others designed by us producing mutations in the TATTTAAA sequence. We also calculated the K D of recombinant E. histolytica TBP (rEhTBP) for several TATA variants. Results E. histolytica nuclear extracts (NE) and TATTT- AAA(1) oligonucleotide form specific complexes Bruchhaus et al. [29] using NE in EMSA and doing in silico analysis proposed that the TATTTAAA sequence is the consensus TATA box for E. histolytica. On the other hand, Luna-Arias et al. [25] showed the homology of EhTBP with human TBP. However, the presence of EhTBP in complexes formed with E. histolytica NE and TATTTAAA(1) oligonucleotide has not been directly demonstrated yet. We first investi- gated the presence of EhTBP in the complex formed by E. histolytica NE and TATTTAAA(1) oligonucleotide by supershift, cross-linking and Western blot assays. When incubated with fresh NE, TATTTAAA(1) oligo- nucleotide migration was retarded, forming a single band (Fig. 1A, lane 1). The NE-TATTTAAA(1) com- plex was specifically competed by TATTTAAA(1) cold oligonucleotide (Fig. 1A, lane 2), whereas it remained when double-stranded poly(dG-dC) or TtTTTttt(7) oligonucleotide were used as unspecific competitors (Fig. 1A, lanes 3 and 4, respectively). The presence of EhTBP in this complex was evidenced in supershift assays by anti-rEhTBP Igs. Two bands appeared when 1 lL of antibodies was added to the mixture (Fig. 1B, lane 3). The lower band comigrated with that formed by NE and TATTTAAA(1) oligonucleotide, whereas the other band migrated slower, due to the partial supershift produced by the antibody. When 5 lL of anti-rEhTBP Igs were added to the mixture, the complex was completely disrupted (Fig. 1B, lane 4), as it has been reported for other supershift experiments [32]. Anti-E. histolytica actin antibodies had no effect on the complex formed (Fig. 1C, lane 2). In cross-linking assays, using a UV-irradiated mix- ture of E. histolytica NE and TATTTAAA(1) oligonu- cleotide, we distinguished a radioactive DNA–protein band of 50 kDa (Fig. 1D, lane 5). This band may be formed by the radioactive probe (11 kDa) bound to endogenous EhTBP (26 kDa) and other protein cross- linked to the complex. As expected, the 50 kDa radio- active band was competed by TATTTAAA(1) cold oligonucleotide (Fig. 1D, lane 6), but it remained in the presence of the poly (dG-dC) unspecific competitor (Fig. 1D lane 7). No complexes were detected in lanes with either nonirradiated or irradiated free probe, or with the nonirradiated oligonucleotide-NE mixture (Fig. 1D, lanes 2–4, respectively). In Western blot assays of UV cross-linked DNA–protein complexes, anti-rEhTBP Igs recognized the radioactive 50 kDa band. This confirms that EhTBP is part of the complex G. de Dios-Bravo et al. Promiscuous E. histolytica TBP FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS 1355 probably associated to another % 13 kDa unknown protein (Fig. 1E, lanes 5–7). The antibodies also recog- nized the same band in the lane where TATTTAAA(1) cold oligonucleotide was used as specific competitor (Fig. 1E, lane 6). As expected, in this lane the complex was formed by the irradiated cold oligonucleotide and NE mixture. Unbound 26 kDa EhTBP comigrated in the gel with the 25 kDa marker (Fig. 1E, lanes 4–7). Data from these experiments altogether demonstrated the presence of EhTBP in NE-TATTTAAA(1) oligo- nucleotide complexes. Recombinant EhTBP binds to TATTTAAA(1) oligonucleotide The rEhTBP was expressed in bacteria as a His 6 - tagged 30 kDa polypeptide. rEhTBP was purified by affinity chromatography and its integrity and identity were verified by Coomassie blue stained gels (SDS ⁄ PAGE) (Fig. 2A) and Western blot assays using anti-rEhTBP Igs (Fig. 2B). In EMSA, purified rEhTBP formed a single band with TATTTAAA(1) probe (Fig. 2C, lane 2). The complex was competed by cold TATTTAAA(1) oligonucleotide, whereas it remained in the presence of poly (dG-dC) unspecific competitor (Fig. 2C, lanes 3 and 4). To discard endogenous TATA binding activity in bacterial extracts, we tested by EMSA the capacity of induced and noninduced bacterial extracts to form complexes with TATTT AAA(1) probe. Results showed that complex was only formed with extracts of induced bacteria expressing rEhTBP (Fig. 2D, lane 3) whereas noninduced bacteria did not form any complexes with TATTTAAA(1) oligonucleotide (Fig. 2D, lane 2). DNA-binding activity of purified rEhTBP for TATA variants In humans and other organisms, variants of the canon- ical TATA box have been reported to be functional [33,34]. On the other hand, the TATTTAAA sequence and several variants are found in many E. histolytica gene promoters at ) 20 to )40 bp upstream the tran- scription initiation site [30], although other TATA variants have been experimentally found at longer distances in Ehtbp and EhRabB genes (our unpublished data). We studied the binding activity of rEhTBP for different TATA sequences present in gene promoters (oligonucleotides TATTTAAA(1), TAT_ _AAA(4), TAT_ _AAg(5) and TATTaAAA(6)), and for mutated versions of TATTTAAA(1) probe [oligonucleotides TAgTgAAA(2) and TATTggAA(3)] (Table 1). We ABCD E Fig. 1. Binding of nuclear extracts to TATTTAAA(1) oligonucleotide. (A) NE (25 lg) and [ 32 P]ATP[cP] end-labeled TATTTAAA(1) oligonucleotide (10 000 c.p.m., 157 p M) were incubated for 15 min at 4 °C for EMSA as described in Experimental procedures. Lane 1, no competitor; lane 2, 300-fold molar excess of unlabeled TATTTAAA(1) oligonucleotide as specific competitor (sc); as unspecific competitors we added 300-fold molar excess of: lane 3, poly(dG-dC) and, lane 4, oligo(dT) 18 . (B) Supershift gel assay using purified anti-rEhTBP Igs. EMSA were performed as above, except that before adding the labeled oligonucleotide, the mixture was preincubated with: lane 1, no NE; lane 2, no antibody; lane 3, 1 lL of purified anti-rEhTBP Igs; lane 4, 5 lL of anti-rEhTBP Igs. (C) Supershift gel assay performed as in B, but using anti-E. histolytica actin Igs. Lane 1, no antibody; lane 2, 5 lL of anti-E. histolytica actin Ig. (D) UV-cross-linking assay of NE (60 lg) and TATTTAAA(1) (50 000 c.p.m., 785 p M). Mixtures for EMSA were UV irradiated at 320 nm for 10 min at 4 °C, analyzed by 12% SDS ⁄ PAGE and radioactivity was determined as described in Experimental procedures. Lane 1, molecular mass markers; lane 2, nonirradiated free probe; lane 3, irradiated free probe; lane 4 , nonirradiated NE-oligonucleotide mixture; lane 5, irradiated NE-oligonucleotide mixture; lane 6, irradiated NE-oligonucleo- tide mixture containing 300-fold molar excess of unlabeled TATTTAAA(1) oligonucleotide as specific competitor (sc); lane 7, irradiated NE-oligonucleotide mixture containing 300-fold molar excess of poly (dG-dC) as unspecific competitor (uc). (E) Western blot assay of UV cross-linked DNA–protein complexes shown in D, using anti-rEhTBP Igs. Promiscuous E. histolytica TBP G. de Dios-Bravo et al. 1356 FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS introduced g’s in the third, fifth and sixth positions of the TATTTAAA(1) sequence, because these positions have been reported as important for DNA-binding activity for human and yeast TBPs [33,34]. DNA-binding activity of rEhTBP for distinct TATA oligonucleotides was evaluated by EMSA using 433 nm (over-saturating concentration) of purified rEhTBP and 10 000 c.p.m. (157 pm) of the probes. Figure 3 displays experiments showing that rEhTBP specifically binds to all oligonucleotides tested. Com- plexes formed by rEhTBP and TATA box variants were fully competed by the same probe and by TAT- TTAAA(1) oligonucleotide (Fig. 3). In these assays, two complexes were observed with TAT_ _AAA(4) and TAT_ _AAg(5) probes, which were specifically competed by TATTTAAA(1) oligonucleotide and by the same probe. The presence of two complexes in some experiments could be due to conformational Table 1. Positions of TATTTAAA (1) sequence and putative TATA variants in E. histolytica gene promoters. TATA variants Gene promoter First nucleotide location b Reference (12 3456 78) a 5’-T A T T T A A A-3’ (1) EhPgp5 Ehactin (-31) c (-30) d [47] (http://www.sanger.ac.uk/Projects/E_histolytica) 5’-T A g T g A A A-3’ (2) Not found 5’-T A T T ggA A-3’ (3) Not found 5’-T A T __A A A-3’ (4) Ehtbp (-109) c (Unpublished) Ehtub1 (-27) d (http://www.sanger.ac.uk/Projects/E_histolytica) EhRabB (-44) c (Unpublished) 5’-T A T __AAg-3’ (5) Ehenol (-50) d (http://www.sanger.ac.uk/Projects/E_histolytica) 5’-T A T T a A A A-3’ (6) Ehpfo (-31) c [48] a Numbers show the base composition in TATA variants. b Nucleotide position is referred to the experimentally c and in silico d determined transcription initiation sites. Putative TATA boxes are defined as TATA sequences upstream of the ATTCA ⁄ G, ATCA or ACGC consensus transcription initiation sites. AB C D Fig. 2. Immunodetection of rEhTBP, and EMSA of TATTTAAA(1) and rEhTBP. rEhTBP was produced by IPTG induced bacteria transformed with the full length Ehtbp gene cloned in pRSET A and purified through nickel NTA-agarose columns as described in Experimental proce- dures. (A) Coomassie blue stained gel (12% SDS ⁄ PAGE) of purified rEhTBP under native conditions. Lane 1, molecular mass markers; lane 2, purified rEhTBP. (B) Western blot assay of purified rEhTBP using anti-rEhTBP Igs. Lane 1, molecular weight markers; lane 2, stripe sequentially incubated with anti-rEhTBP Igs and peroxidase-coupled goat anti-rabbit secondary Igs; lane 3, as in lane 2 but anti-rEhTBP Igs were omitted. (C) EMSA of purified rEhTBP with TATTTAAA(1) oligonucleotide as described in Experimental procedures. Lane 1, free probe; lane 2, no competitor; lane 3, 300-fold molar excess of unlabeled TATTTAAA(1) probe as specific competitor (sc); lane 4, 300-fold molar excess of unspecific competitor (uc). (D) EMSA using 15 lg of bacterial extracts. Lane 1, free probe; lane 2, non induced bacteria (nib) carry- ing pRSET A-Ehtbp plasmid; lane 3, induced bacteria (ib) expressing rEhTBP. G. de Dios-Bravo et al. Promiscuous E. histolytica TBP FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS 1357 changes of the DNA–protein complex, which may affect its electrophoretic migration. To discard the pos- sibility that rEhTBP could bind to any AT rich sequence, we performed a shift assay with rEhTBP and [ 32 P]ATP[cP] end-labeled double stranded TtTTTttt(7), TATaTAtA(8) or TtTTaAAA(9) oligonu- cleotides. rEhTBP did not bind to these sequences (data not shown), indicating that rEhTBP is not merely an AT-rich DNA binding protein without dis- crimination capacity. Obviously, these oligonucleotides did not compete the complex formed with TATTT- AAA(1) and rEhTBP (Fig. 3F). We also verified that in our experiments, rEhTBP was indeed bound to dou- ble-stranded oligonucleotides and not to free labeled single-stranded probes. Labeled probes were passed through a hydroxyapatite column and c.p.m. were counted in the unbound and eluted fractions. In all cases, more than 99% of the radioactivity was found bound to the hydroxyapatite column and it was eluted with 0.4 m phosphate buffer. Figure 3G shows the elu- tion profile for TATTTAAA(1) oligonucleotide as a representative experiment. All together these results showed that rEhTBP has an in vitro binding capacity for distinct TATA elements. Quantification of rEhTBP DNA-binding activity for different TATA oligonucleotides Binding activity of rEhTBP for TATA variants was quantified (as described in Experimental procedures) at A FG BCD E Fig. 3. rEhTBP specifically binds to TATTTAAA(1) oligonucleotide and TATA variants. (A–E) Purified rEhTBP (433 nM) was incubated with [ 32 P]ATP[cP] end-labeled TATA variants (10 000 c.p.m., 157 pM) for EMSA as described in Experimental procedures. Lane 1, free probe; lane 2, no competitor; lane 3, competition with 300-fold molar excess of the same TATA variant as specific competitor (sc); lane 4, competition with 300-fold molar excess of TATTTAAA(1) oligonucleotide; lane 5, competition with 300-fold molar excess of unspecific competitor (uc). The TATA oligonucleotide used in each case is shown below each gel. (F) Control binding assay of rEhTBP (433 n M) with 157 pM (10 000 c.p.m.) of TATTTAAA(1) probe. Lane 1, TATTTAAA(1) free probe; lane 2, purified rEhTBP incubated with TATTTAAA(1) probe; lane 3, purified rEhTBP preincubated with 300-fold molar excess of unlabeled poly (dG-dC) before adding the labeled TATTTAAA(1) probe; lane 4, unlabeled TTTTTTTT(7) oligonucleotide; lane 5, unlabeled TATATATA(8) oligonucleotide, and lane 6 TTTTAAAA(9) oligonucleotide were used as unspe- cific competitors. (G) Elution profile of labeled TATTTAAA (1) probe passed through a hydroxyapatite column as described in Experimental procedures. Fraction 1, unbound single stranded DNA (SS); fractions 2–6, washes with 2.5 mL of 0.12 M phosphate buffer pH 6.8 (W); frac- tions 7–11, elution with 2.5 mL of 0.4 M phosphate buffer pH 6.8 (DS). Volume of each fraction was 0.5 mL. Radioactivity was represented as percentage of the total radioactivity (30 000 c.p.m.) loaded into the column. Promiscuous E. histolytica TBP G. de Dios-Bravo et al. 1358 FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS distinct total rEhTBP concentrations (Fig. 4A–F). Experimental variations for each gel were normalized using the total radioactivity in each lane (complex formed plus free oligonucleotide at the bottom of the gel). Quantification of DNA–protein complexes in each EMSA experiment was performed to calculate K D val- ues of the rEhTBP and each TATA oligonucleotide using the method described by Coleman and Pugh [35] (see Experimental procedures). First, we estimated the c.p.m. present in the shifted protein-TATA oligonucleo- tide (S x ) for each rEhTBP concentration tested (x). Then, the natural logarithms of S x (ln S x ) values were plotted against a given rEhTBP concentration (x). As c.p.m. is a discrete variable, we used ln S x (Eqn 2) to warrant normal distribution of the residual error E in the regression analysis [36,37] in order to obtain more precise and representative data. Thus, these experimen- tal points were fitted as a polynomial function of x (Eqn 2) (Fig. 5). In all cases we obtained a second degree polynomial function describing the relationship between ln S x and x (Table 2). The summary of coeffi- cients, variances, and results of the statistical Student’s t-tests obtained are also presented in Table 2. Once we had defined the mathematical relationship between ln S x and x for each experiment, we deter- mined the average amount of radioactivity (S f ) present in the rEhTBP–TATA oligonucleotide complexes when the reaction reached the titration end point. This was done first using Eqn 3 to calculate the x-value at which the mathematical function has a maximum (x max ). For oligonucleotides TATTTAAA(1), TAgTgAAA(2), TATTggAA(3), TAT_ _AAA(4), and TAT_ _AAg(5), the x max values were 280, 287, 281, 232 and 270 nm, which corresponded to S f values of 324 ± 6, 1038 ± 20, 702 ± 34, 335 ± 10, 431 ± 9 c.p.m., respectively. In the case of TATTaAAA(6) oligonucleotide, which formed two specific DNA–protein complexes, the x max values were 261 and 307 nm for the slower and faster bands, respectively, which corresponded to 190 ± 5 and 325 ± 4 c.p.m. The next step was to obtain F-values using Eqn 1 as described in Experimental procedures and plot it ver- sus the rEhTBP ⁄ TATA molar ratio. F-values also fit- ted to a polynomial function of the molar ratio of rEhTBP ⁄ TATA oligonucleotide. Figure 6A shows an example of the F experimental points obtained for TAT_ _AAg(5) oligonucleotide. Results for all oligo- nucleotides showed a second degree polynomial func- tion (Table 2). The statistical test gave similar results to those obtained for Eqn 2. Then, we obtained the rEhTBP ⁄ TATA oligonucleotide molar ratios at which F corresponds to 1 (maximum value). rEhTBP ⁄ TATA oligonucleotide molar ratios were 1458, 1959, 1599, 2006 and 2030 for TATTTAAA(1), TAgTgAAA(2), TATTggAA(3), TAT_ _AAA(4), and TAT_ _AAg(5) ABC DEF Fig. 4. Affinity quantification of rEhTBP-TATA variant complexes as a function of the rEhTBP concentration by EMSA. (A–F) EMSA of [ 32 P] ATP[cP] end-labeled TATA variants (10 000 c.p.m., 157 p M) incubated with different rEhTBP concentrations as described in Experimental pro- cedures: lane 1, 0 n M; lane 2, 50 nM; lane 3, 97 nM; lane 4, 145 nM; lane 5, 193 nM; lane 6, 242 nM; lane 7, 290 nM, and lane 8, 338 n M. Arrows show the complexes analyzed. The TATA oligonucleotide used in each case is shown below each gel. G. de Dios-Bravo et al. Promiscuous E. histolytica TBP FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS 1359 Fig. 5. Graphical representation of data obtained in quantification of rEhTBP-TATA variant complexes. (A–F) ln of S x (the radioactivity present in DNA–protein complexes) versus x (rEhTBP concentrations). Data were obtained from EMSA experiments as shown in Fig. 4. F-1 and F-2 correspond to the slower and faster DNA–protein complexes formed with TATTaAAA(6) oligonucleotide, respectively. Dots represent experi- mental data. Continuous line is the graph predicted by the second degree polynomial function (Eqn 2 in Experimental procedures). The TATA oligonucleotide used in each case is shown below each graph. Table 2. Mathematical relationships between ln S x vs. x and ln F vs. rEhTBP ⁄ TATA molar ratio. a, Coefficients of the equation ln Sx ¼ a 0 + a 1 x+a 2 x 2 +E) N(0,1) and ln F ¼ a 0 +a 1 (rEhTBP ⁄ TATA) + a 2 (rEhTBP ⁄ TATA)2 + E ) N(0,1). Sa is the standard deviation of a n coefficients. I, slower DNA-protein complex; II, faster DNA-protein complex. ln S x vs. x ln F vs. rEHTBP ⁄ TATA molar ratio a 0 a 1 a 2 S 2 a 0 S 2 a 1 S 2 a 2 a 0 a 1 a 2 S 2 a 0 S 2 a 1 S 2 a 2 1 5.85 7.66 x 10 )3 )1.34 · 10 )5 1.20 · 10 )1 1.48 · 10 )6 6.01 · 10 )12 )1.10 1.12 · 10 )3 )2.86 · 10 )7 1.20 · 10 )1 3.16 · 10 )8 2.76 · 10 )15 2 4.37 1.56 · 10 )2 )2.77 · 10 )5 8.24 · 10 )1 1.25 · 10 )5 6.31 · 10 )11 )2.19 2.74 · 10 )5 )8.56 · 10 )7 8.24 · 10 )1 3.87 · 10 )7 6.03 · 10 )14 3 5.04 7.91 · 10 )3 )1.16 · 10 )5 1.68 · 10 )1 2.55 · 10 )6 1.29 · 10 )11 )1.34 1.24 · 10 )3 )2.87 · 10 )7 1.68 · 10 )1 6.29 · 10 )8 7.80 · 10 )15 4 5.48 4.34 · 10 )3 )8.03 · 10 )6 1.96 · 10 )1 2.98 · 10 )6 1.50 · 10 )11 )5.87 5.78 · 10 )4 )1.42 · 10 )7 1.96 · 10 )1 5.30 · 10 )8 4.72 · 10 )15 5 3.39 1.42 · 10 )2 )2.73 · 10 )5 2.44 · 10 )1 3.71 · 10 )6 1.87 · 10 )11 )1.86 2.23 · 10 )3 )6.71 · 10 )7 2.44 · 10 )1 9.13 · 10 )8 1.13 · 10 )14 6I 4.61 7.62 · 10 )3 )1.24 · 10 )5 6.54 · 10 )2 9.94 · 10 )7 5.01 · 10 )12 )1.17 1.46 · 10 )3 )4.57 · 10 )7 6.54 · 10 )2 3.67 · 10 )8 6.82 · 10 )15 6II 4.76 7.28 · 10 )3 )1.30 · 10 )5 1.16 · 10 )1 1.76 · 10 )6 8.85 · 10 )12 )1.02 1.40 · 10 )3 )4.80 · 10 )7 1.16 · 10 )1 6.48 · 10 )8 1.20 · 10 )14 Promiscuous E. histolytica TBP G. de Dios-Bravo et al. 1360 FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS oligonucleotides, respectively. For the complexes formed with TATTaAAA(6) oligonucleotide, the val- ues were 1664 and 1599 for the slower and faster bands, respectively. The reciprocal of all these values were then used to calculate the total active rEhTBP concentrations (P T ) (see Experimental procedures) as reported [35]. K D values of rEhTBP for TATA variants The reciprocal of F-values gave the following linear function: (1 ⁄ F) ¼ 1+K D (1 ⁄ P). The slope of this lin- ear function corresponds to K D . Therefore, the data (1 ⁄ F) and (1 ⁄ P) were fitted using the robust linear regression method [36,37], which should give an equa- tion of the type (1 ⁄ F) ¼ c 0 + c 1 (1 ⁄ P) if the variables were linearly related. An example of the fitness between these variables using the robust linear regression method is presented for TAT_ _AAg(5) oligonucleo- tide (Fig. 6B). These calculations were performed for all TATA oligonucleotides. K D values and their standard deviations are shown in Table 3. K D values of rEhTBP for TATA variants ranged between 1.04 (± 0.39) · 10 )11 and 1.60 (± 0.37) · 10 )10 m, which corresponded to oligonucleo- tides TAT_ _AAA(4) and TAgTgAAA(2), respectively. TATTTAAA(1) and TAT_ _AAA(4) oligonucleotides had the lowest K D values that did not significantly dif- fer each other (Table 3). Additionally, oligonucleotides TATTggAA(3) and the two complexes formed with TATTaAAA(6) gave similar K D values. The next larger value corresponded to TAT_ _AAg(5), and the largest to TAgTgAAA(2). Therefore, we could order the oligonucleotides according to their TBP affinity as follows: TATTTAAA(1) ¼ TAT_ _AAA(4) > TATTgg AA(3) ¼ TATTaAAA(6) > TAT_ _AAg(5) > TAgTg AAA(2). Discussion In this paper we studied the rEhTBP affinity for sev- eral TATA variants present in E. histolytica gene pro- moters and TATA box versions designed by us (Table 1). Our data showed that the promiscuity of rE- hTBP for TATA variants is higher than those reported for Homo sapiens, Saccharomyces cerevisiae and Ara- bidopsis thaliana TBPs [33,38]. Therefore, in addition to TATTTAAA(1) sequence, we showed here that TAT_ _AAA(4), TAT_ _AAg(5) and TATTaAAA(6) are, at least in vitro, EhTBP binding motifs. In addi- tion, rEhTBP can also bind in vitro to TAgTgAAA(2) and TATTggAA(3) oligonucleotides that are mutated versions of TATTTAAA(1) sequence. Thus, based on our in vitro experiments, the E. histolytica TATA box could be proposed as 5¢-(1: T)(2: A)(3: T ⁄ G)(4: T ⁄ G ⁄ A)(5: T ⁄ G ⁄ A)(6: A ⁄ G)(7: A) (8: A)-3¢ (numbers indicate the nucleotide position in TATA box). In vitro transcription assays are needed to accurately establish Fig. 6. Relationships between ln F and rEhTBP ⁄ TAT_ _AAg(5) molar ratio, and 1 ⁄ F and 1 ⁄ P. (A) Experimental data obtained from relationships between ln F and rEhTBP ⁄ TAT_ _AAg(5) molar ratio. (B) Graphical representation of 1 ⁄ F and 1 ⁄ P for the rEhTBP ⁄ TAT_ _AAg(5) complexes. The dots represent experimentally obtained data. The continuous line is the graph predicted by the second degree polynomial function (A) and linear function for K D estimation (B). Table 3. Dissociation constants of rEhTBP for TATA variants. Oligonucleotide (K D ± SD) M 5’-TATTTAAA-3’ (1) 1.96 (± 0.58) · 10 -11 5’-TAgTgAAA-3’ (2) 1.60 (± 0.37) · 10 -10 5’-TATTggAA-3’ (3) 3.18 (± 1.16) · 10 -11 5’-TAT_ _AAA-3’ (4) 1.04 (± 0.39) · 10 -11 5’-TAT_ _AAg-3’ (5) 8.26 (± 2.20) · 10 -11 5’-TATTaAAA -3’ (6) 4.28 (± 0.47) · 10 -11 a 3.94 (± 0.44) · 10 -11 b a Upper DNA-protein complex; b lower DNA-protein complex; SD, Standard deviation. G. de Dios-Bravo et al. Promiscuous E. histolytica TBP FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS 1361 the quantitative value of each base in different position and in vivo experiments will demonstrate the function of these TATA elements in the cell. Based on a systematic X-ray crystallographic study of the A. thaliana TBP isoform 2, Patikoglou et al. [38] defined the TATA sequence as an eight bp variable motif formed by 5¢-T ) c>a¼ g ⁄ A ) t ⁄ T ) a ¼ c ⁄ A ) t ⁄ T ) a ⁄ A ) g>c¼ t ⁄ A ¼ T>g>c⁄ G ¼ A>c¼ t-3¢. Recently, in S. cerevisiae, Basehoar et al. [39] identified the TATA box element as TAT A(A ⁄ T)A(A ⁄ T)(A ⁄ G). A. thaliana TBP isoform 2 recognizes 10 variants of the adenovirus major late pro- moter TATA element that in many organisms is located at )25 to )40 bp from the transcription initi- ation site. However, some S. cerevisiae gene promoters have the TATA box at )40 to )120 bp [13,40] and the Ehtbp gene presents the TAT_ _ AAA sequence located at )109 bp from the transcription initiation site that, accordingly to our unpublished data, might func- tion as TATA element. Additionally, recent results gave also evidence that the EhRabB gene promoter TATA box maps at )44 bp (Rodrı ´ guez, M.A., personal communication) (Table 1). In about 20 E. histolytica genes, the transcription ini- tiation site is known [29] and from these data the TAT TTAAA sequence has been proposed as the canonical EhTBP binding motif. However, this is the first published report experimentally demonstrating that rEhTBP binds to TATTTAAA and other related sequences. Here, we experimentally showed that EhTBP is in the complex formed in vitro by the consen- sus TATTTAAA(1) oligonucleotide and NE (Fig. 1) and that rEhTBP specifically binds to this DNA sequence (Fig. 2). However, E. histolytica genes contain different TATA elements (Table 1) that could be used by TFIID transcription factor. We also showed that rEhTBP forms specific complexes with all TATA vari- ants tested, although with different affinity, showing a more relaxed DNA-binding specificity of EhTBP than those described for other systems [33,34,38,41]. DNA-binding activities of H. sapiens [34,38], A. thali- ana [38] and S. cerevisiae [33,34] TBPs are severely affec- ted when TATA oligonucleotides contain g’s in first, second, third, fourth, fifth and sixth positions. In con- trast, EhTBP formed complexes with TAgTgAAA(2) and TATTggAA(3) oligonucleotides used here (Fig. 3), indicating that g’s in these positions do not affect EhTBP DNA-binding activity, and showing that at least in in vitro assays, EhTBP is even more promiscuous than other TBPs studied. In vivo studies are needed to define whether this also occurs in the trophozoites. The dip in data at higher titration point in curves of Figs 5 and 6 can be explained by the dimerization [35] or oligomerization [42] of TBP molecules at high TBP concentration. These multimers have no ability to bind DNA [35,42]. We cannot discard multiple TBP binding events. K D values of rEhTBP for TATA variants ranged from 10 )11 to 10 )10 m (Table 3). These results indica- ted that: (1) the rEhTBP has similar affinity for TAT TTAAA(1) and TAT_ _AAA(4) oligonucleotides (2) variations of the nucleotide at position 5 slightly reduce the affinity of rEhTBP for TATA variant in relation to the TATTTAAA oligonucleotide; (3) TAT_ _AAg(5) and TAgTgAAA(2) oligonucleo- tides are bound by rEhTBP with less affinity than those TATA variants designed by us. An alignment of EhTBP with the H. sapiens, A. thaliana and S. cerevisiae TBPs showed that EhTBP has the residues reported as involved in TATA box binding in the same positions than other TBPs [27,38]. Thirty of them are identical (Fig. 7, open arrowheads) and of the remaining seven residues, five are conserved and two are nonconserved changes (Fig. 7, filled arrowheads). Interestingly, EhTBP presents a T in position 192, which corresponds to V203 in yeast, to V161 in A. thaliana and to V301 in human TBPs (Fig. 7, arrow). Strubin and Struhl [34] substituted the V203 of yeast TBP by T and the resultant mutant TBP showed an increased DNA-binding activity for the TGTAAA element of the his3 gene promoter. Thus, the presence of T192 in EhTBP sequence could influ- ence its DNA-binding specificity for TATA variants. However, this is still to be experimentally demonstra- ted. The promiscuous DNA-binding activity of EhTBP may have conferred an evolutionary advantage to E. histolytica, because certain mutations in the TATA box would not affect gene expression. Experimental procedures E. histolytica cultures Trophozoites of E. histolytica clone A (strain HM1:IMSS) [1] were axenically cultured in TYI-S-33 medium at 37 °C and harvested during exponential growth phase [43]. Electrophoretic mobility shift assays (EMSA), competitions and supershift gel assays using NE and rEhTBP Aliquots of 25 lg of NE, obtained as described [23], or 30– 300 ng (50–500 nm) of purified rEhTBP [25] were used for EMSA. NE or rEhTBP were incubated for 15 min at 4 °C with poly(dG-dC) (1 lgÆlL )1 ) in binding buffer containing 12 mm Hepes pH 7.9, 60 mm KCl, 10% (v ⁄ v) glycerol and Promiscuous E. histolytica TBP G. de Dios-Bravo et al. 1362 FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS 1mm each dithiothreitol, EDTA, spermidine and MgCl 2 . Then, [ 32 P]ATP[cP] end-labeled double-stranded oligo- nucleotides (10 000 c.p.m., 157 pm): 5¢-TATTTAAA-3¢(1), 5¢-TAgTgAAA-3¢(2), 5¢-TATTggAA-3¢(3), 5¢-TAT_ _AAA- 3¢(4), 5¢-TAT_ _AAg-3¢(5), 5¢-TATTaAAA-3¢(6) (Table 1), 5¢-TtTTTttt-3¢(7), 5¢-TATaTAtA-3¢(8) or 5¢-TtTTaAAA- 3¢(9) were added to the mixture. Incubation continued for other 10 min at 4 °C. Mutations introduced in TATTT AAA box are marked in small letters and deletions are in dashes in the oligonucleotide sequences. In all cases, flank- ing bases were added to obtain 18 bp length oligonucleo- tides. Numbers in parenthesis after the sequences identify each oligonucleotide. For supershift gel assays, before add- ing oligonucleotides, the mixture was preincubated for 15 min at 4 °C with 1 or 5 lL of purified rabbit anti- rEhTBP Igs [25] or with 5 lL of anti-E. histolytica actin Igs (kindly given by Manuel Herna ´ ndez, CINVESTAV, IPN, Me ´ xico). In competition experiments, 300-fold molar excess of tested oligonucleotide, or the TATTTAAA(1) oligo- nucleotide, or the unspecific competitors poly(dG-dC), TtTTTttt(7), TATaTAtA(8) or TtTTaAAA(9) were incuba- ted with the mixture 10 min before the probe was added. Samples were electrophoresed on 6% nondenaturing polyacrylamide gels (PAGE) in 0.5X TBE. Gels were vacuum-dried and radioactive complexes were detected in a Phosphor Imager apparatus (Bio-Rad). Shifted radioactive bands were quantified by densitometry using the Quantity One software version 4 (Bio-Rad). All experiments reported here were carried out at least three times by duplicate with reproducible results. To discard the presence of single-stran- ded oligonucleotides, labeled probes (30 000 c.p.m.) were passed through a hydroxyapatite (Bio-Rad) column (1 cm in length · 0.7 cm in diameter) at 4 °C. We collected 0.5 mL fractions. The flowthrough contained the unbound material, which corresponds to single stranded DNA. The column was washed with 2.5 mL of 0.12 m phosphate buf- fer pH 6.8 and double-stranded DNA was eluted with 2.5 mL of 0.4 m phosphate buffer pH 6.8 [44]. Finally, radioactivity in each fraction was measured in a Beckman LS 6500 liquid scintillation counter. Cross-linking and Western blot assays Protein concentration of NE was measured by the Bradford method [45]. For cross-linking assays, 60 lg of proteins were incubated with radioactive TATTTAAA(1) oligonucle- otide (50 000 c.p.m., 785 pm) and UV irradiated at 320 nm directly on a transilluminator apparatus (UVP Inc., San Gabriel, CA, USA) for 10 min at 4 °C. Complexes formed after cross-linking assays were resolved through 12% SDS ⁄ PAGE. Gels were scanned in a Phosphor Imager apparatus and were transferred to nitrocellulose membranes for Western blot assays [46]. Membranes were blocked with 0.05% (v ⁄ v) Tween 20 and 5% (w ⁄ v) nonfat milk in NaCl ⁄ P i for 2 h at room temperature and incubated over- night at 4 °C with purified anti-rEhTBP Igs (1 : 1000). Im- munoreactivity was detected with peroxidase-labeled goat anti-rabbit Igs (Zymed, San Francisco, CA, USA) (1 : 2000) and chemiluminescence method, using ECL Plus Kit (Amersham, Piscataway, NJ, USA). Expression and purification of rEhTBP and anti-rEhTBP Igs generation The full-length Ehtbp gene, cloned in pRSET A [25] was expressed in Escherichia coli BL21(DE3)pLysS strain (Invitrogen, Carlsbad, CA, USA) as a His 6 -tagged 30 kDa polypeptide. Proteins from IPTG (1 mm) induced bacteria were separated by 12% SDS ⁄ PAGE and gels were Fig. 7. Predicted amino acid residues involved in EhTBP binding to DNA. Conserved C-terminal domain sequences of TBPs from Saccharomycces cerevisiae (ScTBP) (P13393), Arabidopsis thaliana (ArathTBP2) (P28148), Homo sapiens (hTBP) (P20226) and E. histolytica (EhTBP) (P52653) were aligned using the CLUSTAL W program. Black boxes indicate identical amino acids in at least two sequences and grey boxes the amino acid conserved changes. Unfilled and filled arrowheads indicate the 37 amino acid residues involved in DNA binding activity. 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Entamoeba histolytica TATA- box binding protein binds to different TATA variants in vitro Guadalupe de Dios-Bravo 1,2 ,. probably due to modifications in amino acids involved in TBP-DNA binding. Abbreviations EhTBP, Entamoeba histolytica TATA- box binding protein; EMSA, electrophoretic