We studied the binding activity of rEhTBP for different TATA sequences present in gene promoters oligonucleotides TATTTAAA1, TAT_ _AAA4, TAT_ _AAg5 and TATTaAAA6, and for mutated version
Trang 1to 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-Camarillo2and 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
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; 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 KDvalues 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.
Trang 2transcription 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
KD 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
Trang 3probably 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 His6
-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
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 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) oligonucleoNE-oligonucleo-tide 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.
Trang 4introduced 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.
(1 2 3 4 5 6 7 8)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 g g A A-3’ (3) Not found
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.
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) expresscarry-ing rEhTBP.
Trang 5changes 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 [32P]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
Fig 3 rEhTBP specifically binds to TATTTAAA(1) oligonucleotide and TATA variants (A–E) Purified rEhTBP (433 n M ) was incubated with [ 32 P]ATP[cP] end-labeled TATA variants (10 000 c.p.m., 157 p M ) 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 p M (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.
Trang 6distinct 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 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 Sxand 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 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 (xmax) 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
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)
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 n M ; lane 3, 97 n M ; lane 4, 145 n M ; lane 5, 193 n M ; lane 6, 242 n M ; lane 7, 290 n M , and lane 8, 338 n M Arrows show the complexes analyzed The TATA oligonucleotide used in each case is shown below each gel.
Trang 7Fig 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 Experiexperi-mental 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.
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
Trang 8oligonucleotides, 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 (PT) (see Experimental procedures) as
reported [35]
KDvalues of rEhTBP for TATA variants
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
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
KD values and their standard deviations are shown
in Table 3 KD values of rEhTBP for TATA variants ranged between 1.04 (± 0.39)· 10)11 and 1.60 (± 0.37)· 10)10m, which corresponded to oligonucleo-tides TAT_ _AAA(4) and TAgTgAAA(2), respectively TATTTAAA(1) and TAT_ _AAA(4) oligonucleotides had the lowest KDvalues 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)
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 KD
estimation (B).
Table 3 Dissociation constants of rEhTBP for TATA variants.
3.94 (± 0.44) · 10 -11 b
a Upper DNA-protein complex; b lower DNA-protein complex; SD, Standard deviation.
Trang 9the 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 > a ¼ g ⁄ A t ⁄ T a ¼
c⁄ A t ⁄ T a ⁄ A g > c ¼ 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 Addifunc-tionally, 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
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) 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 thalianaand 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 37C 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 4C
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
Trang 101 mm each dithiothreitol, EDTA, spermidine and MgCl2.
Then, [32P]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 4C 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 4C 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 4C 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⁄ Pifor 2 h at room temperature and incubated over-night at 4C 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 His6-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.
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.