Genomic structure of the human TATA-box-binding protein (TBP)

Gene, 161 (1995) 2177282 0 1995 Elsevier Science B.V. All rights reserved.

277

037%1119/95/$09.50

GENE 08927

Genomic structure of the human TATA-box-binding protein (TBP) (Intron/exon;

Christian

promoter; HeLa cells; internal donor site; phage k vector; transcription factor)

Chalut *, Yves Gallois *, Arnaud

lnstitut de GtWtique et de Biologie MolCculaire et Cellulaire, Received by C.M. Kane: 8 December

Poterszman,

Vincent Moncollin

BP 163, 67404 Illkirch Ctde.y C.L;. de Strasbourg,

1994; Revised/Accepted:

9 February,/11

February

and Jean-Marc

Egly

France

1995; Received at publishers:

16 March

1995

SUMMARY

The gene encoding the human TATA-box-binding protein (hTBP) is contained within a 20-kb DNA fragment and is split into eight exons. The coding sequence is interrupted by six introns and the S-untranslated region (5’-UTR) of the gene by a 2.5-kb intron. A comparison of the hTBP exon/intron organization with the various TBP cloned to date is presented.

INTRODUCTION

The TBP factor, originally defined as the TATA-boxbinding protein, is required for transcription directed not only by RNA polymerase II from TATA-containing and TATA-less promoters, but is also necessary for genes transcribed by RNA polymerase I and III when included in a multisubunit complex called TFIID, SLl and TFIIIB, respectively (for a review, see Moncollin et al., 1994 and references therein). The TBP cDNAs that have been cloned from various organisms reveal a highly conserved C-terminal domain, spanning 180 aa. in addition to a non-conserved N-terminal domain the length of which shows some Correspondence to: Dr. J.-M. Egly, Institut de Genetique et de Biologie Moleculaire et Cellulaire, 1 rue Laurent Fries, BP 163, 67404 Illkirch Ctdex,

CU.

de Strasbourg,

France.

Tel. (33-88)

653-447;

Fax (33-88)

653-201; e-mail: [email protected] *These authors Abbreviations:

should

be considered

A., ilcanthamoeba;

TBP; bp, base pair(s);

cDNA,

equal contributors.

aa, amino acid(s); aTBP, A. castellanii DNA complementary

to RNA; hTBP,

human TBP; kb, kilobase or 1000 bp; mTBP, mouse TBP; N (n), A or C or G or T(U); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading G; S., Saccharomyces;

frame; PCR, polymerase SDS, sodium

TBP; Sz, Schizosaccharomyces;

dodecyl

chain reaction;

R (r), A or

sulfate; spTBP,

Sz. pombe

TBP, TATA-box-binding

gene (DNA, RNA) encoding TBP; tsp, transcription UTR, untranslated region(s); Y (y), C or T(U). SSDI 037X-l

119(95)00209-X

species specific variability. The crystal structure of the Arabidopsis TBP-2 (Nikolov et al., 1992) and of the C-terminal domain of the yeast TBP (Chasman et al., 1993) revealed a structure resembling a saddle which interacts with the minor groove of the DNA (Starr and Hawley, 1991; Lee et al., 1991) hence inducing a bending of the promoter DNA around the TATA-box region (Horikoshi et al., 1992; Kim, J.L. et al., 1993; Kim, Y. et al., 1993). To address the question of how the TBP gene is regulated and whether the conserved C-terminal domain within the two direct repeats (Cavallini et al., 1989) originate from some conserved genomic structure, we have studied the genomic organization of the hTBP. We show that the coding sequence is split into seven exons and the 5’-UTR of the gene is interrupted by a 2.5-kb intron. Several A + T-rich sequences located upstream from this intron are candidates for the TATA-box sequence. The genomic structure we have characterized was compared with those of the various TBP genes cloned to date.

protein; start

TBP.

point(s);

EXPERIMENTAL

AND DISCUSSION

(a) Structure of the human TBP gene

To isolate genomic hTBP clones, a human placenta genomic library was screened by using the hTBP cDNA

278 and an oligo (Probe a, Figs. lb and 2) derived from the 5’-UTR of the hTBP cDNA as probes. Two positive clones (hh41 and hh3P; Fig. la) were isolated and were aligned with respect to the hTBP cDNA by restriction mapping and Southern blot hybridization. hh41 was sequenced and shown to contain the complete hTBP cDNA ORF, as well as part of the S’JJTR and the entire 3’-UTR, whereas hh3P contains the first four coding exons and the S-UTR hTBP. The coding sequence of hTBP is contained within a 20-kb fragment and is split into seven exons (numbered II to VIII in Fig. lb) of 205, 440, 88, 92, 168, 95 and 664 bp in length, respectively (Figs. lb and 2). The sequence corresponding to the 5’-end of the hTBP cDNA was localized 2.5 kb upstream from the first translated exon (Fig. 2). The sequences at the exon/intron junctions (Fig. 2 and Table I) are in agreement with the consensus sequences for splicing donor (S-AG/GTRAGT-3’) and acceptor sites (5’-Y,NYAG/G-3’) (Breathnach and Chambon, 1981), except for the exon III/intron junction.

(a)

Polypyrimidine tracts associated with sequences close to the branch-point consensus sequence (YNYURAY; reviewed in Smith et al., 1989) are present upstream from the 3’-splice acceptor site of each intron/exon boundary. The ATG start (Met) codon is located in exon II and is preceded by a large 5’-UTR ( 151 nt). This exon encodes the first 18 aa (Fig. 2). The last exon (exon VIII) encodes the 25 aa of the C-terminal region of the protein, as well as the entire 583-bp 3’-UTR of the cDNA. The polyadenylation signal (S-AATAAA) is present 569 nt downstream from the stop codon (at position +649 of exon VIII). Sequence comparison with the previously cloned hTBP cDNA (Hoffmann et al., 1990a; Kao et al., 1990; Peterson et al., 1990) shows that at position +94 of exon VI, a T is replaced by a C although this causes no change in the predicted aa sequence. In addition, the isolated TBP gene encodes 37 consecutive Gln (aa 58-94), corroborating with genetic studies which have shown that the number of Gln is highly polymorphic in the human population (Imbert et al., 1994).

i

I

pJ

kh41

1 hh3P

BumHI

Fjj

I

I

I

I

I

I I3

I

II

III

IV

V

VI

VII VIII

I

I

(

I

EcoRI XbaI Hind111

I

I

I

I

I

I

I-N-terminal

(b)

I

I

domain 7

Conserved I

C-terminal

domain

basic core

165

195

1

o homolo&

225

281

313

338

Protein

i

I\

S$

s

,\

,f?

II

/’ ,’

:

‘\ 1 ‘ \

’

,’

DNA

I

/’ I’

,,T ,’ ,’ ,’

I

I’

:: ’ ,I ‘\ $1 \ ,I \

looI&

genomic

i

;

’ ’ ’

’

:

,

,’

: ,’ : ,’

t ’

”

I

I

I’:

: ,’ : : ,’ : ,’

;1

,:

I’I I’ : I’ ;

,’ ,’

:

:

I

,’

:

::

:,

’ : ’

,’

I :

,;

:I

1

;

: : : I 1’ I ;

poly@)

r----

-152

I

494

54

II

III

582

614

IV

V

842

VI

937

VII

1014

1602

VIII

Fig. 1. Organization of hTBP. (a) Schematic representation of the hh41 and hh3P clones. Exons I-VIII (see Table I) are indicated. Bent arrow represents tsp. (b) Complete exon/intron organization of the hTBP. UZ'R(hatched boxes) and coding regions (blackened boxes) are indicated relative to the protein sequence (Met at position 1) and to the coding sequence of TEP (A residue of the ATG start codon at position + 1). Probe a, derived from the 5'-UTRof the hTBP cDNA, used for library screening is indicated, as well as the putative TATA boxes (triangles) and the polyadenylation site. The two repeats, the polyglutamine sequence (PolyQ), the o homology and the basic core are also represented. Methods: A human placenta genomic library

constructed

in hGEM-12

(a kind gift of J.M. Garnier),

was screened

by plaque hybridization

(50% formamide/

solution/l% SDS/100 pg sperm DNA salmon at 42°C overnight; washing with 2 x SSC/O.l% SDS at 42°C and 0.2 x hTBP cDNA which was 32P-labeled by random priming (Feinberg and Volgelstein, 1983) and with Probe a. Phage positive clones (Grossberger, 1987; Dumanski et al., 1988) and used for restriction enzyme mapping. Fragments of the in the pBluescript SK(+) (Stratagene, La Jolla, CA, USA) and regions containing the exons were sequenced on both termination method (Sanger et al., 1977). SSC is 0.15 M NaCI/O.OlS M Na,.citrate pH 7.

x SSC/l x Denhardt’s

SSC/O.l% SDS at 42°C) of the DNA was extracted from the positive clones were subcloned strands by the dideoxy chain-

Fig. 2. The nt sequence are presented.

of the hTBP and deduced

The bold sequences

the hTBP cDNA (cDNA). (Pr. Ext.) are indicated determined

the beginning

by arrows.

by restriction

aa sequence.

in the 3’- and 5’-liTR

the putative

DNA fragments

TATA boxes and the polyadenylation

et al., 1990) and the two putative

of oligos a, b, c, d is also indicated.

on the genomic

as well as part of the 3’- and 5’- ends of the intron

consensus

of the hTBP cDNA poly(A) (Peterson

L,ocation

site analysis

Exona I--VIII (boxed),

indicate

Asterisk

subcloned

tspdetermined

(*) marks the stop codon.

into pBluescript

SK(+)

Methods:

and confirmed

sequences

signal. The 5’-end of by primer extension Intron

sizes have been

by PCR analysis

as described

elsewhere (Sambrook et al., 1989). In order to confirm the presence of an additional intron in the 5’-UTR, a reverse PCR was performed on HeLa and using M-MLV reverse cell total RNA. First-strand cDNA synthesis was carried out by priming HeLa cell total RNA ( 15 pg) with an ohgo(d transcriptase cloning

TABLE

(Superscript,

in pBluescript

BRL) and was used for PCR amplification

SK(+).

Primer

extension

was carried

with oligos b and c as primers.

out as described

by Sambrook

The amplified

fragment

was sequenced

after

et al. (1989) using oligo d.

I

Introniexon

junctions

of the hTBP”

Exon

S-Splice

donor AG

No.

gtragt-

3’-Splice accept01

Intron

aa

Y,,nyag

size

interrupted

(kb)

by intron

G-

Size (bpl -ACCCfi

qtaa_ca-

ccaaag

CAGCAT-

2.5

II

205

-CCTCA(;

gtaata-

ccacaq

GGTGCC-

4.85

Gln'*/Gly'"

III

440

-GCTGCA

qtqaqt-

ttacaq

AAATAT-

2.35

Gln'65

IV

xx

-CCCA&

ctaa-

ttqtaq

CGGTTT-

2.4

Lys'94/Arg'95

V

Y2

-CAAG&

qtagcc-ccctaq

TGAAGA-

2.5

SeP

VI

16X

-TAGT&

qtaaqt-

ttctaq

-TAAC&

gtaaqt-

tcttaq

TTATGA(;TGCTA-

1.55 0.7

Se?' G]yJ13

‘,

I

VII VIII a Upper donor

and lower case letters represent and acceptor

sites (indicated

exon and intron

in the heading

sequences,

to column

respectively.

Nucleotides

which lit with the consensus

sequences

for splicing

2) are underlined.

(b) Characterization of the 5’-UTR of the hTBP Southern blot analysis and restriction mapping performed on the genomic clone hh3P showed that the sequence corresponding to the -230 to - 152 upstream region of the hTBP cDNA is found 2.65 kb upstream

from the start codon (data not shown). Sequence analysis of this 2.65-kb fragment confirmed that this clone contained the sequence corresponding to the -230 to - 152 upstream region of the cDNA sequence, thus indicating that the 5’-UTR of the hTBP is interrupted by a 2.5-kb

280 intron (intron 1) located 151 nt upstream from the start codon (Fig. 2). This was further confirmed by a reverse PCR performed on HeLa cell total RNA using oligo b and oligo c (Fig. 2). The amplified fragment was 376 nt in length and sequencing analysis showed that it contained the exonI/exonII junction sequence. These results demonstrate unambiguously the existence of another exon (exon I) in front of a 2.5-kb intron (intron 1). In order to define the 5’-end of the hTPB mRNA, in vitro transcription assays using hTBP DNA-fragments located upstream to intron 1 (Fig. 2) (Chalut et al., 1994) and RACE (Rapid Amplification cDNA Ends) PCR were performed, although no further information could be obtained by either method. A primer extension performed on total HeLa cell RNA by using oligo d resulted in two major signals corresponding to DNA fragments of 180 and 400 nt (Fig. 2). The putative tsp corresponding to the larger signal is preceded by a A + T-rich region which could be used as a TATA box (Fig. 2). According to Northern blot analysis, the sizes of the TBP mRNA varies from 1.8 to 2.2 kb, which implies that the tsp should be present 200-600 nt upstream from the ATG start codon (50-450 nt upstream from the 3’-end of exon I). This fits perfectly with our primer extension results. However, we were able to amplify the region located upstream to the putative tsp by reverse-PCR experiments on total HeLa cell RNA, thus demonstrating that the promoter region is located in a more upstream region (data not shown). Two consensus TATA-boxes were identified 842 and 643 nt upstream from the 3’-end of exon I (Fig. 2). If one of these could be considered as the promoter, it would result in a mRNA of 2.55 and 2.35 kb, respectively, slightly longer than that detected by Northern blot analysis. Our inability to localize the hTBP tsp strongly suggests that either the promoter is located in a more upstream region that could include an additional intron

or that such a gene may possess a weak promoter under the controls of enhancer sequences not yet identified. (c) Comparison of the TBP structure between various species Alignment of the TBP cDNAs cloned from several organisms reveal that the protein could be divided into two domains (Hoffmann et al., 1990a). We attempted to correlate these different structures with the intron-exon organization of the mouse, A. castellanii and Sz. pombe TBP genes (Sumita et al., 1993; Hoffmann et al., 1990b; Wong et al., 1992). The S. cerevisiae TBP has no intron. The mouse TBP (mTBP) gene was found to extend over 18 kb and contains seven coding exons, whereas the Sz. pombe (spTBP) and A. castellanii (aTBP) TBP (1.67 and 1.23 kb, respectively) contain three and two introns, respectively. The N-terminal domain is exclusively contained within one exon in Sz. pombe and A. castellaneii and in two exons in mouse and human (Fig. 3). However, the location of the intron in the N-terminal domain of hTBP and m7’BP are different: This intron is inserted at nt 54 of the coding sequence in hTBP (between exons II and III) but at nt 36 in mTBP (between exons I and II) (position 1 corresponds to the A base of the first Met codon; Fig. 3). In the mouse, this intron is much shorter in size (1.2 vs. 4.85 kb; Sumita et al., 1993). The conserved C-terminal domain of TBP, which contains the two direct repeats, is split by five introns in both mouse and human (Fig. 3). Their locations are the same in mTBP and hTBP but their size differs. In the spTBP and aTBP, this conserved domain contains three and two introns, respectively. Intron 6 of the hTBP interrupts the coding sequence at 1-bp difference compared to intron 3 of the spTBP and intron 2 of the aTBP. Locations of intron 3 and intron 4 of the hTBP are the same as the

Human

Sz pombe

Fig. 3. Schematic representation of the TBP structures in various species. The boxes correspond to the aa sequence encoded by each exon and are numbered by roman numerals for each species. Arabic numbers between each box indicate the intron number for each species. Those aa interrupted by introns are indicated at the extremities of the exons. The white and black colored parts correspond to the variable N-terminal domain and to the conserved C-terminal domain, respectively, of each TBP protein. Hatched boxes represent PolyQ regions. Locations of the two repeats are represented by arrows.

281 locations of intron 1 of the aTBP and intron 2 of spTBP. respectively. In higher eukaryotes (human and mouse), the two direct repeats are separated by a single intron (intron 5 and 4 for human and mouse, respectively). whereas such separations do not occur in A. castelaneii and SZ. pombe. Both repeats are interrupted by an intron in the mouse and human genes, whereas the first repeat of A. castelaneii is not interrupted and the first repeat of Sz. pombe is interrupted by two introns. The sequence encoding for the first 8 aa of the C-terminal domain, which is not included in the repeat sequence, belongs to a different exon (exon III in human and exon II in mice), which encodes most of the N-terminal region. This coding region is also located in a different exon in the aTBP and spTBP. (d) Correlation with tertiary structure of the protein

As the crystallographic structure of the TATA-boxbinding polypeptide has been determined (Nikolov et al.. 1992; Chasman et al., 1993), it is possible to locate the exon borders on the 3D structure of the protein and to analyze their distribution. Exons IV and V of the hTBP have approximately the same length and their sequences encode the aa which belong to the first repeat (aa 165224). The sequence encoding the second repeat (aa 2555315) is located within exons VI (aa 255-281) VII (aa 2822313) and partially VIII (aa 314-315). Exon VI also encodes the protein region (aa 225-254) which separates the two direct repeats. Alignment of the two repeats

shows that their C-terminal domains are highly conserved (Fig. 4a). These conserved domains are encoded by exons V and VII, while several of the residues therein have been conserved among diverse species throughout evolution (Nikolov and Burley, 1994) and are implicated in DNA binding (Kim and Burley. 1994: Fig. 4b) Although there is not a rigorous correspondence between exons and structural elements of the protein, a certain correlation can be seen (Fig. 4b). lntrons tend to be located at the extremities of surface loops or at the end of secondary structure elements such as b-strands. Apart from intron 3, located in near the pseudo-2-fold axis, introns 4 and 5 are located within regions of the gene encoding loops whereas introns 6 and 7 match the end of a b-strand. Residues from exons V and VII are related by the pseudo-two fold axis. Interestingly. these residues correspond to the center strands of the repeat (strands S3, S4. S5 and S3’, S4’. SS’).

(e) Conclusions

Taken together, this study represents a major contribution to the understanding of the organization of the hTBP gene. Due to its universal character, the organization of the TBP gene in various species could provide a good model for the study of the origin of introns. Our present data may help to clarify this matter which is still subject to controversy.

(a) ExonIll

,

Exon

IV

Exon

I

I

Exon VI exon

Exon VII

V

Exon

I

VI

Exon VIII

I

III exon

_

VI

exon VII

(b) exon

VI

exon

VII

Fig. 4. Structure of TBP. (a) Alignment of the two repeats of the hTBP. represent aa which belong to the repeats. Two asterisks and one asterisk

exon

exon

IV

V

Only aa of the C-termmal conserved domain are shown. Bold characters (*) indicate identical aa and similar aa, respectively. Open circles indicate

aa interacting with the DNA (Kim and Burley, 1994). (b) Three-dimensional representation of the exonic structure from TBP. The ribbon model of the TATA-box binding protein, drawn using the atomic coordinates from the X-ray structure of the S. cerevisiae TBP (Chasman et al., 1993) has been shaded according the exonic structure of the human gene: exons III. V and VII in black, exons IV, VI and VIII in light grey.

282 ACKNOWLEDGEMENTS

We thank P. Chambon for his continuous support. We also thank R. Roy and J.C. Thierry for critical reading of the manuscript and fruitful discussions. This work was supported by funds from the Association pour la recherche contre le Cancer (A.R.C) and the Centre National de la Recherche Scientifique (C.N.R.S.). C.C. was supported by a fellowship from the A.R.C.

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lmbert, G., Trottier, Y., Beckmann, J. and Mandel, J.L.: The gene for the TATA binding protein (TBP) that contains a highly polymorphic protein coding CAG repeat maps to 6q27. Genomics 21 (1994) 6677668. Kao, C.C., Lieberman, P.M., Schmidt, M.C., Zhou, Q, Pei, R. and Berk, A.J.: Cloning of a transcriptionally active human TATA binding factor. Science 248 (1990) 1646-1649. Kim, J.L. and Burley, SK.: 1.9 A resolution refined structure of TBP recognizing the minor groove of TATAAAAG. Struct. Biol. 1 (1994) 638653. Kim, J.L., Nikolov, D.B. and Burley, S.K.: Co-crystal structure of TBP recognizing the minor groove of a TATA element. Nature 365 (1993) 520-527. Kim, Y., Geiger, J.H., Hahn, S. and Sigler, P.B.: Crystal structure of a yeast TBP/TATA-box complex. Nature 365 (1993) 512.-520. Lee, D.K., Horikoshi, M. and Roeder, R.G.: Interaction of TFIID in the minor groove of the TATA element. Cell 67 (1991) 1241-1250. Moncollin, V., Roy, R. and Egly, J.M.: The TATA saga: structure and function of the TATA-binding factor. In: Conaway. R.C. and Conaway, J.W. (Eds.), Transcription: Mechanisms and Regulation. Raven Press, New York, NY, 1994, pp. 45-62. Nikolov, D.B., Hu, S-H., Lin, J., Gasch, A., Hoffmann, A., Horikoshi, M., Chua, N.-H., Roeder, R.G. and Burley, S.K.: Crystal structure of TFIID TATA-box binding protein. Nature 360 (1992) 40-46. Nikolov, D.B. and Burley, S.K.: 2.1 A resolution relined structure of a TATA box-binding protein (TBP). Struct. Biol. 1 (1994) 621-637. Peterson, M.G., Tanese, N., Pugh, B.F. and Tjian, R.: Functional domains and upstream activation properties of cloned human TATA binding protein. Science 248 (1990) 162551630. Sambrook, J., Maniatis, T. and Fritsch, E.F.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 546335467. Smith, C.W.J., Patton, J.G. and Nadal-Ginard, B.: Alternative splicing in the control of gene expression. Annu. Rev. Genet. 23 (1989) 527-571. Starr, D.B. and Hawley, D.K.: TFIID binds in the minor groove of the TATA box. Cell 67 (1991) 123ll1240. Sumita, K., Makino, Y., Katoh, K., Kishimoto, T., Muramatsu, M., Mikoshiba, K. and Tamura, T.: Structure of a mammalian TBP (TATA-binding protein) gene: isolation of the mouse TBP genome. Nucleic Acids Res. 21 (1993) 2769. Wong, J.M., Liu, F. and Bateman, E.: Isolation of genomic DNA encoding transcription factor TFIID from Acanthamoeba castellnnii: characterization of the promoter. Nucleic Acids Res. 20 (1992) 481774824.