Báo cáo khoa học: Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro

Entamoeba histolytica TATA-box binding protein binds to different TATA variants in vitro Guadalupe de Dios-Bravo1,2, Juan Pedro Luna-Arias3, Ana Marı´a Rivero´ n4, Jose´ J Olivares-Trejo5, Ce´ sar Lo´ pez-Camarillo2 and Esther Orozco5

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

Keywords Entamoeba histolytica; KD; promiscuous DNA-binding activity; TATA-binding protein; TATA variants

Correspondence Esther Orozco, Departamento de Patologı´a Experimental, Centro de Investigacio´ n y de 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 KD 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.

functions

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 [11–13]. Mechanisms, molecules and DNA sequences controlling the spatial and temporal

Abbreviations EhTBP, Entamoeba histolytica TATA-box binding protein; EMSA, electrophoretic mobility shift assays; rEhTBP, recombinant Entamoeba histolytica TATA-box binding protein; NE, nuclear extracts.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1354

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

Results

E. histolytica nuclear extracts (NE) and TATTT- AAA(1) oligonucleotide form speciﬁc complexes

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.

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).

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 its biological activity has been poorly [27,28], but studied.

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

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 KD of recombinant E. histolytica TBP (rEhTBP) for several TATA variants.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1355

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

A

B

C

D

E

Fig. 1. Binding of nuclear extracts to TATTTAAA(1) oligonucleotide. (A) NE (25 lg) and [32P]ATP[cP] end-labeled TATTTAAA(1) oligonucleotide (10 000 c.p.m., 157 pM) were incubated for 15 min at 4 (cid:1)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 pM). Mixtures for EMSA were UV irradiated at 320 nm for 10 min at 4 (cid:1)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.

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 form any complexes with TATTTAAA(1) did not oligonucleotide (Fig. 2D, lane 2).

probably associated to another (cid:1) 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.

DNA-binding activity of puriﬁed rEhTBP for TATA variants

Recombinant EhTBP binds to TATTTAAA(1) oligonucleotide

veriﬁed

by Coomassie

stained

blue

The rEhTBP was expressed in bacteria as a His6- tagged 30 kDa polypeptide. rEhTBP was puriﬁed by afﬁnity chromatography and its integrity and identity were 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 lanes 3 and 4). To discard endogenous (Fig. 2C,

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 (Table 1). We TAgTgAAA(2) and TATTggAA(3)]

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1356

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

A

B

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.

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 locationb

Reference

(1 2 3 4 5 6 7 8)a

5’-T A T T T A A A-3’ (1)

[47] (http://www.sanger.ac.uk/Projects/E_histolytica)

(-31)c (-30)d

5’-T A g T g A A A-3’ (2) 5’-T A T T g g A A-3’ (3) 5’-T A T _ _ A A A-3’ (4)

5’-T A T _ _ A A g-3’ (5) 5’-T A T T a A A A-3’ (6)

EhPgp5 Ehactin Not found Not found Ehtbp Ehtub1 EhRabB Ehenol Ehpfo

(-109)c (-27)d (-44)c (-50)d (-31)c

(Unpublished) (http://www.sanger.ac.uk/Projects/E_histolytica) (Unpublished) (http://www.sanger.ac.uk/Projects/E_histolytica) [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.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1357

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

A

B

C

D

E

F

G

Fig. 3. rEhTBP speciﬁcally binds to TATTTAAA(1) oligonucleotide and TATA variants. (A–E) Puriﬁed rEhTBP (433 nM) was incubated with [32P]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 nM) 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.

double

[32P]ATP[cP]

shown),

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

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 stranded end-labeled and TtTTTttt(7), TATaTAtA(8) or TtTTaAAA(9) oligonu- cleotides. rEhTBP did not bind to these sequences (data not 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

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1358

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

total

(Fig. 4A–F). rEhTBP concentrations distinct 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).

the reaction reached the titration end point. This was done ﬁrst using Eqn 3 to calculate the x-value at which function has a maximum (xmax). the mathematical For oligonucleotides TATTTAAA(1), TAgTgAAA(2), TATTggAA(3), TAT_ _AAA(4), and TAT_ _AAg(5), the xmax values were 280, 287, 281, 232 and 270 nm, which corresponded to Sf 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 xmax values were 261 and 307 nm for the slower and faster bands, respectively, which corresponded to 190 ± 5 and 325 ± 4 c.p.m.

Quantiﬁcation of DNA–protein complexes in each EMSA experiment was performed to calculate KD 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 (Sx) for each rEhTBP concentration tested (x). Then, the natural logarithms of Sx (ln Sx) values were plotted against a given rEhTBP concentration (x). As c.p.m. is a discrete variable, we used ln Sx (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 Sx 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 Sx and x for each experiment, we deter- mined the average amount of radioactivity (Sf) present in the rEhTBP–TATA oligonucleotide complexes when

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)

A

B

C

D

E

F

Fig. 4. Afﬁnity quantiﬁcation of rEhTBP-TATA variant complexes as a function of the rEhTBP concentration by EMSA. (A–F) EMSA of [32P] ATP[cP] end-labeled TATA variants (10 000 c.p.m., 157 pM) incubated with different rEhTBP concentrations as described in Experimental pro- cedures: lane 1, 0 nM; 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 nM. Arrows show the complexes analyzed. The TATA oligonucleotide used in each case is shown below each gel.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1359

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

Fig. 5. Graphical representation of data obtained in quantiﬁcation of rEhTBP-TATA variant complexes. (A–F) ln of Sx (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 Sx vs. x and ln F vs. rEhTBP ⁄ TATA molar ratio. a, Coefﬁcients of the equation ln Sx ¼ a0 + a1x + a2x2 + E ) N(0,1) and ln F ¼ a0 + a1(rEhTBP ⁄ TATA) + a2(rEhTBP ⁄ TATA)2 + E ) N(0,1). Sa is the standard deviation of an coefﬁcients. I, slower DNA-protein complex; II, faster DNA-protein complex.

ln F vs. rEHTBP ⁄ TATA molar ratio

ln Sx vs. x

S2a0

S2a1

S2a2

S2a0

S2a1

S2a2

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 1 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 2 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 3 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 4 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 5 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

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1360

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

Table 3. Dissociation constants of rEhTBP for TATA variants.

Oligonucleotide

(KD ± SD) M

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)

1.96 (± 0.58) · 10-11 1.60 (± 0.37) · 10-10 3.18 (± 1.16) · 10-11 1.04 (± 0.39) · 10-11 8.26 (± 2.20) · 10-11 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.

method is presented for TAT_ _AAg(5) oligonucleo- tide (Fig. 6B). These calculations were performed for all TATA oligonucleotides.

and

between

1.04 (± 0.39) · 10)11

KD values and their standard deviations are shown in Table 3. KD values of rEhTBP for TATA variants 1.60 ranged (± 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 KD 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 KD 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).

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 the rEhTBP ⁄ representation of 1 ⁄ F and 1 ⁄ P for 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 KD estimation (B).

Discussion

the

oligonucleotides, complexes respectively. For 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 (PT) (see Experimental procedures) as reported [35].

KD values of rEhTBP for TATA variants

in vitro experiments,

(8: A)-3¢

The reciprocal of F-values gave the following linear function: (1 ⁄ F) ¼ 1 + KD (1 ⁄ P). The slope of this lin- ear function corresponds to KD. 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 c0 + c1 (1 ⁄ P) if the variables were linearly related. An example of the ﬁtness between regression these variables using the robust

linear

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 the E. histolytica TATA our box could be proposed as 5¢-(1: T)(2: A)(3: T ⁄ G)(4: (numbers T ⁄ G ⁄ A)(5: T ⁄ G ⁄ A)(6: A ⁄ G)(7: A) indicate the nucleotide position in TATA box). In vitro transcription assays are needed to accurately establish

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1361

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

the quantitative value of each base in different position and in vivo experiments will demonstrate the function of these TATA elements in the cell.

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.

in many organisms

KD 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) the nucleotide at position 5 slightly variations of 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.

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 (cid:2) c > a ¼ g ⁄ A (cid:2) t ⁄ T (cid:2) a ¼ c ⁄ A (cid:2) t ⁄ T (cid:2) a ⁄ A (cid:2) g > c ¼ t ⁄ A ¼ T > g > c ⁄ G ¼ A > c ¼ 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- is that moter TATA element 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).

is

this

to TATTTAAA and other

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

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, the ﬁrst published report experimentally demonstrating that rEhTBP binds related experimentally showed that sequences. Here, we 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].

Trophozoites of E. histolytica clone A (strain HM1:IMSS) [1] were axenically cultured in TYI-S-33 medium at 37 (cid:1)C and harvested during exponential growth phase [43].

Electrophoretic mobility shift assays (EMSA), competitions and supershift gel assays using NE and rEhTBP

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.

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 (cid:1)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

The dip in data at higher titration point in curves of Figs 5 and 6 can be explained by the dimerization [35]

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1362

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

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. Filled arrowheads correspond to the seven amino acid residues involved in DNA binding activity that are changed in EhTBP sequence. Arrow denotes the amino acid change in EhTBP position 192.

[32P]ATP[cP]

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

Igs

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 (cid:1)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 ⁄ Pi for 2 h at room temperature and incubated over- night at 4 (cid:1)C with puriﬁed anti-rEhTBP Igs (1 : 1000). Im- munoreactivity was detected with peroxidase-labeled goat anti-rabbit (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

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1363

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 His6-tagged 30 kDa polypeptide. Proteins from IPTG (1 mm) induced bacteria separated by 12% SDS ⁄ PAGE and gels were were 1 mm each dithiothreitol, EDTA, spermidine and MgCl2. end-labeled double-stranded oligo- Then, 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 (cid:1)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 (cid:1)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- the unspeciﬁc competitors poly(dG-dC), nucleotide, or TtTTTttt(7), TATaTAtA(8) or TtTTaAAA(9) were incuba- ted with the mixture 10 min before the probe was added. electrophoresed on 6% nondenaturing Samples were in 0.5X TBE. Gels were polyacrylamide gels (PAGE) 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 (cid:1)C. We collected 0.5 mL fractions. The ﬂowthrough contained the unbound material, which corresponds to single stranded DNA. The

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

coefﬁcient an. We took the difference (an ) 0) as signiﬁcant only if the Student’s t-test probability P(t) was lower than 0.05.

Once we estimated the coefﬁcients of Eqn 2 for each experiment, we determined the rEhTBP concentration xmax at which the curve reached a maximum by deriving (d) Eqn 2 and solving it for zero value (Eqn 3).

ðEqn 3Þ dðln SxÞ=dðxÞ ¼ 0

Coomassie blue stained. rEhTBP was puriﬁed by nickel- agarose afﬁnity columns as described by the manufacturer (Qiagen, Standford, CA, USA). Then, 150 lg of puriﬁed rEhTBP were subcutaneously inoculated three times in rabbits each 15 days. One week after last immunization, rabbits were bled. Antibodies were twice precipitated from serum with 60% (w ⁄ v) (NH4)2SO4, dialyzed using NaCl ⁄ Pi buffer and immunoadsorbed against nitrocellulose-immobi- lized rEhTBP before using them for supershift and Western blot assays.

Then, we used xmax value in Eqn 2 to obtain the Sf value. Finally, F was calculated with Eqn 1 for each rEhTBP con- centration x.

Quantiﬁcation of DNA–protein complexes

Estimation of the molar ratio of rEhTBP ⁄ TATA oligonucleotide when F ¼ 1

Complexes formed by distinct probes with rEhTPB were quantiﬁed by densitometry measuring pixels in bands by the quantity one software and normalized against free probe to correct the total c.p.m. loaded in each lane of the gel.

Determination of dissociation constants (KD) of DNA–protein complexes

The KD for EhTBP and each TATA oligonucleotide was determined by EMSA as described [35] with some modiﬁca- tions. Complexes formation with rEhTBP and radiolabeled oligonucleotides was measured as a function of protein con- centration. The fraction (F) of DNA probe bound by pro- teins was calculated using the Eqn 1

ðEqn 1Þ F ¼ ðSx (cid:3) S0Þ=ðSf (cid:3) S0Þ

Following the method described by Coleman and Pugh [35], we estimated the fraction of active rEhTBP that binds to TATA oligonucleotides (active unbound plus active bound rEhTBP), assuming a binding stoichiometry of 1. Therefore, F was plotted as a function of the molar ratio of total rEhTBP to TATA oligonucleotides. As for Eqn 2, we ﬁtted ln F as a polynomial function of the molar ratio of rEhTBP ⁄ TATA oligonucleotide. The ﬁtness was also done by the least square method and the maximum of the curve was determined as before. When F ¼ 1 (the saturating point), the reciprocal of the x intercept was multiplied by total rEhTBP concentration to get the total fraction of act- ive rEhTBP in the mixture. We called it (x ⁄ TATA)max, which according to Coleman and Pugh [35] it corresponds to F ¼ 1.

Conﬁdence intervals (CI) of polynomial function predictions

where, Sx is the radioactivity present in the shifted protein- DNA complex at protein concentration x. S0 is the corres- ponding radioactivity present when x ¼ 0 (i.e. the absence of protein). Sf is the average amount of radioactivity present when F becomes independent of x (i.e. when the reaction rea- ches the titration end point).

Conﬁdence intervals were estimated using Yp ± CI, in which Yp is the predicted value by the function for a particular x, and CI is deﬁned by Eqn 4

ðEqn 4Þ CI ¼ ½1=tð0:975; glÞ(cid:5)f½X0R(cid:3)1X(cid:5)r2(cid:5)1=2 To accurately determine Sf values for each EMSA experi- ment we plotted the Sx values for each rEhTBP concentra- tion tested (x). Then, we determined the polynomial function best describing the curve behavior as:

ðEqn 2Þ ln Sx ¼ a0 þ a1x þ a2x2 þ (cid:4) (cid:4) (cid:4) þ anxn þ E (cid:3) Nð0; 1Þ

where t(0.975, gl) is the Student’s t-test for 0.975 percentile, and gl the degrees of freedom; X¢, is the vector of the val- ues raised to the transposed X vector, R)1 is the inverse of the regression matrix, and r2 is the residual variance.

KD calculation

The KD of protein–DNA complexes was calculated from Eqn 5 [35]

ðEqn 5Þ F ¼ P=ðKD þ PÞ

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1364

where, P is the uncomplexed active rEhTBP. P is related to total active rEhTBP concentration PT by Eqn 6 where ln Sx is the natural logarithm of Sx, an is the numer- ical coefﬁcient, n is the equation degree and E is the resid- ual error in the regression analysis [36,37]. As c.p.m. is a discrete variable, we used ln Sx (Eqn 2) to warrant normal distribution of the residual error E in the regression analy- sis [36,37]. The ﬁtness analysis was performed by least square regression analysis. The degree of Eqn 2 that we determined in our experiments was n ¼ 2, that was the power of x that corresponded to the last coefﬁcient differ- ing signiﬁcantly from zero. We estimated the statistical Stu- dent’s t-test (t) for each an as tn ¼ (an )0) ⁄ Saj, where j ranges from 0 to n, and San is the standard deviation of the

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

(Eqn 6Þ

PT ¼ P þ PD

8 Que X & Reed SL (2000) Cysteine proteinases and the pathogenesis of amebiasis. Clin Microbiol Rev 13, 196– 206. 9 Bruchhaus I, Loftus BJ, Hall N & Tannich E (2003) where, PD is the rEhTBP concentration in the protein– DNA complex. The apparent association equilibrium con- stant Ka is the reciprocal of KD.

The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation. Eukaryot Cell 2, 501–509. 10 Zhai Y & Saier MH Jr (2000) The amoebapore super- family. Biochem Biophys Acta 1469, 87–99. 11 Hahn S (1998) The role of TAFs in RNA polymerase II transcription. Cell 95, 579–582. As Eqn 5 is a hyperbolic function, then 1 ⁄ F should ﬁt to a linear function of 1 ⁄ P and therefore KD is the slope of this line. These two variables were ﬁtted by means of a robust regression method [36,37] that avoided the deleterious effect of data outliers on KD values. This ﬁtness does not assume normal distribution of residual error. Calculations of coefﬁ- cients and variances were done by programming iterative algorithms which used least square estimates as initial values. 12 Berk AJ (1999) Activation of RNA polymerase II tran- scription. Curr Opin Cell Biol 11, 330–335. 13 Smale ST & Kadonaga JT (2003) The RNA polymerase

Acknowledgements

II core promoter. Annu Rev Biochem 72, 449–479. 14 Lee TI & Young RA (2000) Transcription of eukaryotic

protein-coding genes. Annu Rev Genet 34, 77–137. 15 Levine M & Tjian R (2003) Transcription regulation and animal diversity. Nature 424, 147–151.

This work was supported by CONACYT (Me´ xico) and by the European Community. We are grateful to Mr Alfredo Padilla-Barberi for his excellent technical assistance in the artwork.

16 Burley SK & Roeder RG (1996) Biochemistry and

References

structural biology of transcription factor IID (TFIID). Annu Rev Biochem 65, 769–799.

17 Hochheimer A & Tjian R (2003) Diversiﬁed transcrip- tion initiation complexes expand promoter selectivity and tissue-speciﬁc gene expression. Genes Dev 17, 1309– 1320. 1 Orozco E, Sua´ rez ME & Sa´ nchez T (1985) Differences in adhesion, phagocytosis and virulence of clones from Entamoeba histolytica, strain HM1: IMSS. Int J Parasi- tol 15, 655–660. 18 Hernandez N (1993) TBP, a universal eukaryotic tran- scription factor? Genes Dev 7, 1291–1308.

2 De Menezes Feitosa L, Salgado LM, Rodrı´ guez MA, Vargas MA & Orozco E (1997) Phenotype variability and genetic polymorphism in Entamoeba histolytica clo- nal populations. Arch Med Res 28, 27–29. 19 Imbalzano AN, Zaret KS & Kingston RE (1994) Facili- tated binding of TATA-binding protein to nucleosomal DNA. J Biol Chem 18, 8280–8286.

20 Bucher P (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences. J Mol Biol 212, 563–568. 3 Padilla-Vaca F, Ankri S, Bracha R, Koole LA & Mirel- man D (1999) Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal ⁄ GalNAc lectin. Infect Immun 67, 2096–2102.

4 Bruchhaus I, Roeder T, Lotter H, Schwerdtfeger M & Tannich E (2002) Differential gene expression in Enta- moeba histolytica isolated from amoebic liver abscess. Mol Microbiol 44, 1063–1072. 21 Gilchrist CA, Holm CF, Hughes MA, Schaenman JM, Mann BJ & Petri WA Jr (2001) Identiﬁcation and char- acterization of an Entamoeba histolytica upstream regu- latory element 3 sequence-speciﬁc DNA-binding protein containing EF-hand motifs. J Biol Chem 276, 11838– 11843. 22 Schaenman JM, Gilchrist CA, Mann BJ & Petri WA Jr

5 Petri WA Jr, Smith RD, Schlesinger PH, Murphy CF & Ravdin JI (1987) Isolation of the galactose-binding lec- tin that mediates the in vitro adherence of Entamoeba histolytica. J Clin Invest 80, 1238–1244.

(2001) Identiﬁcation of two Entamoeba histolytica sequence-speciﬁc URE4 enhancer-binding proteins with homology to the RNA-binding motif RRM. J Biol Chem 276, 1602–1609. 23 Go´ mez C, Pe´ rez DG, Lo´ pez-Bayghen E & Orozco E 6 Tannich E, Ebert F & Horstman RD (1991) Primary structure of the 170-kDa surface lectin of pathogenic Entamoeba histolytica. Proc Natl Acad Sc USA 88, 1849–1853.

(1998) Transcriptional analysis of the EhPgp1 promoter of Entamoeba histolytica multidrug-resistant mutant. J Biol Chem 273, 7277–7284.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1365

24 Marchat LA, Go´ mez C, Pe´ rez DG, Paz F, Mendoza L & Orozco E (2002) Two CCAAT ⁄ enhancer binding protein sites are cis-activator elements of the Entamoeba 7 Garcı´ a-Rivera G, Rodrı´ guez MA, Oca´ diz R, Martı´ nez- Lo´ pez MC, Arroyo R, Gonza´ lez-Robles A & Orozco E (1999) Entamoeba histolytica: a novel cysteine protease and an adhesin form the 112 kDa surface protein. Mol Microbiol 33, 556–568.

G. de Dios-Bravo et al.

Promiscuous E. histolytica TBP

Silverman, BW, eds), pp. 13–15. Oxford University Press, Oxford, UK. histolytica EhPgp1 (mdr-like) gene expression. Cell Microbiol 4, 725–737. 25 Luna-Arias JP, Herna´ ndez-Rivas R, de Dios-Bravo G, 37 Lopez-Canovas L, Biscay R, Noa MD, PerezPerez G,

Herrera JA, Orozco E & Riveron AM (1998) Compari- son of DNA migration in two CHEF chambers of dif- ferent sizes. J Chromat 806, 187–197. Garcı´ a J, Mendoza L & Orozco E (1999) The TATA-box binding protein of Entamoeba histolytica: cloning of the gene and location of the protein by immunoﬂuorescence and confocal microscopy. Microbiol 145, 33–40.

38 Patikoglou GA, Kim JL, Sun L, Yang SH, Kodadek T & Burley SK (1999) TATA element recognition by the TATA box-binding protein has been conserved through- out evolution. Genes Dev 13, 3217–3230. 39 Basehoar AD, Zanton SA & Pugh F (2004) Identiﬁca-

tion and distinct regulation of yeast TATA box-contain- ing genes. Cell 116, 699–700.

26 Mendoza L, Orozco E, Rodrı´ guez MA, Garcı´ a-Rivera G, Sa´ nchez T, Garcı´ a E & Gariglio P (2003) Ehp53, an Entamoeba histolytica protein, ancestor of the mamma- lian tumour suppressor p53. Microbiol 149, 885–893. 27 Nikolov DB, Chen H, Halay ED, Hoffmann A, Roeder RG & Burley SK (1996) Crystal structure of a human TATA box-binding protein ⁄ TATA element complex. Proc Natl Acad Sci USA 93, 4862–4867.

40 Chen W & Struhl K (1988) Saturation mutagenesis of a yeast his3 ‘TATA element’: genetic evidence for a speci- ﬁc TATA-binding protein. Proc Natl Acad Sci USA 85, 2691–2695.

28 De Dios-Bravo G, Lo´ pez C, Luna-Arias JP & Orozco E (2000) DNA binding activity and predicted tertiary structure of the TATA binding protein of Entamoeba histolytica. Arch Med Res 31, S299–S300.

41 Hoopes BC, LeBlanc JF & Hawley DK (1992) Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway. J Biol Chem 16, 11539– 11547.

29 Bruchhaus I, Leippe M, Lioutas C & Tannich E (1993) Unusual gene organization in the protozoan parasite Entamoeba histolytica. DNA Cell Biol 12, 925–933. 30 Purdy JE, Pho LT, Mann BJ & Petri WA Jr (1996)

Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin. Mol Biochem Parasitol 78, 91–103. 42 Perez-Howard GM, Weil PA & Beechem JM (1995) Yeast TATA binding protein interaction with DNA: Fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics. Biochemistry 34, 8005–8017. 31 Singh U, Gilchrist CA, Schaenman JM, Rogers JB, 43 Diamond LS, Harlow R & Cunnick C (1978) A new

medium for the axenic cultivation of Entamoeba histoly- tica and other Entamoeba. Trans R Soc Trop Med Hyg 72, 431–432. 44 Richard EJ (1994) Separation of double- and Hockensmith JW, Mann BJ & Petri WA Jr (2002) Con- text-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly. Mol Biochem Parasitol 120, 107–116.

single-stranded nucleic acids using hydroxylapaytite chromatography. In Current Protocols in Molecular Biology (Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Sheidman, JG, Smith, JA & Struhl, K, eds), pp. 2.13.1–2.13.3. John Wiley & Sons, Inc., Chichester, UK. 32 Bidder M, Shao JS, Charlton-Kachigian N, Loewy AP, Semenkovich CF & Towler DA (2002) Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities. J Biol Chem 277, 44485–44496. 33 Wobbe CR & Struhl K (1990) Yeast and human

45 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util- izing the principle of protein-dye binding. Anal Biochem 72, 248–254. 46 Towbin H, Staehelin T & Gordon J (1979) Electro- TATA-binding proteins have nearly identical DNA sequence requirements for transcription in vitro. Mol Cell Biol 10, 3859–3867.

34 Strubin M & Struhl K (1992) Yeast and human TFIID with altered DNA-binding speciﬁcity for TATA ele- ments. Cell 68, 721–730. phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4356. 35 Coleman RA & Pugh BF (1995) Evidence for func- 47 Pe´ rez DG, Go´ mez C, Lo´ pez-Bayghen E, Tannich E &

tional binding and stable sliding of the TATA binding protein on nonspeciﬁc DNA. J Biol Chem 270, 13850–13859. Orozco E (1998) Transcriptional analysis of the EhPgp5 promoter of Entamoeba histolytica multidrug-resistant mutant. J Biol Chem 273, 7285–7292.

FEBS Journal 272 (2005) 1354–1366 ª 2005 FEBS

1366

36 Atkinson AC (1985) The algebra of deletion. In Plots, Transformations and Regression: an Introduction to Graphical Methods of Diagnostic Regression Analysis (Copas, JB, Dawid, AP, Eagleson, GK, Pierce, DA & 48 Rodrı´ guez MA, Hidalgo ME, Sa´ nchez T & Orozco E (1996) Cloning and characterization of the Entamoeba histolytica pyruvate: ferredoxin oxidoreductase gene. Mol Biochem Parasitol 78, 273–277.