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. 1998 Apr;180(7):1771–1776. doi: 10.1128/jb.180.7.1771-1776.1998

The TATA-Binding Protein (TBP) from the Human Fungal Pathogen Candida albicans Can Complement Defects in Human and Yeast TBPs

Ping Leng 1, Philip E Carter 2, Alistair J P Brown 1,*
PMCID: PMC107089  PMID: 9537374

Abstract

Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed the C. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation in Saccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

Candida albicans is the major fungal pathogen in humans, causing irritating superficial candidoses, as well as life-threatening systemic infections (34, 35). The fungus is capable of a reversible morphological transition between yeast and hyphal growth forms, and it can undergo phenotypic switching at high frequencies among a variety of colony morphologies. Both morphogenesis and phenotypic switching probably contribute to the pathogenicity of C. albicans.

The general transcription initiation factor TBP (TATA-binding protein) plays a key role in the activation and regulation of eukaryotic promoters. The binding of TBP to the TATA element initiates the assembly of a preinitiation complex involving RNA polymerase II and other general initiation factors (4, 8, 25, 33). The stimulation of transcription by different trans-acting factors is mediated through distinct TATA elements, suggesting that different types of TBP complex may exist in yeasts (7, 18, 46), humans (43, 44, 52, 53), and plants (27). TBP-associated factors are not generally required for transcriptional activation in yeast (31). Nevertheless, they might act as molecular bridges between different activators and the general transcription machinery (40). Although transcriptional regulation probably plays a critical role in a number of developmental and adaptive responses in C. albicans, little is known about transcriptional control in this fungus.

To facilitate the study of interactions between sequence-specific transcription factors and the preinitiation complex in C. albicans, we have isolated and characterized the C. albicans TBP gene (TBP1). We show that the single TBP1 locus in C. albicans encodes a functional TBP with significant C-terminal sequence similarity to TBPs from diverse organisms.

MATERIALS AND METHODS

Growth, morphogenesis, and sporulation.

Saccharomyces cerevisiae and C. albicans strains were grown in YPD or SD (41). Hyphal growth of C. albicans was induced with serum at 37°C, as described previously (49), and analyzed after 105 min. Yeast tetrad analysis was performed by using standard techniques (42) on the S. cerevisiae strain YAB668 (his3/his3, SPT15/spt15::HIS3) (13) transformed (16, 23) either with the C. albicans TBP1 genomic clone or with the parental vector (YCp50).

Library screening and DNA sequencing.

C. albicans TBP1 cDNA clones were isolated by amplifying S. cerevisiae SPT15 (encoding TBP [17]) by PCR and by using the 0.72-kb PCR product to screen a C. albicans Lambda ZAP II cDNA library (47) by plaque hybridization under low-stringency conditions (37). SPT15 PCR primers were 5′-ATGGCCGATGAGGAACGTTTA-3′ and 5′-TCACATTTTTCTAAATTCACTTAG-3′.

TBP1 genomic clones were isolated from a C. albicans genomic library (45) generously provided by M. Payton (Glaxo Institute for Molecular Biology, Geneva, Switzerland). Colony hybridization (37) was performed with the 1.4-kb C. albicans TBP1 cDNA as a probe.

TBP1 cDNA and genomic clones were sequenced to completion (38) with Dye Terminator cycle sequencing kits (Perkin-Elmer, Worington, United Kingdom) and run on an ABI 377 automated DNA sequencer. DNA sequences were analyzed with the Genetics Computer Group programs (11) on the Biotechnology and Biological Sciences Research Council computer at the Daresbury Laboratory.

Filter hybridizations.

Genomic DNA was prepared from C. albicans 3153 (30) and subjected to Southern blot analysis (37) by standard procedures. For Northern blot analysis (37), total RNA was prepared from C. albicans (3), electrophoresed on agarose-formaldehyde gels, blotted onto Hybond nylon membranes, and hybridized with the 1.4-kb C. albicans TBP1 cDNA radiolabelled by random priming (12). mRNA levels were quantified by phosphorimaging with a Bio-Rad GS-525. Signals were measured relative to those of the rRNAs by loading equal amounts of total RNA in the lanes of the Northern blot gels, because there has been no report so far of a suitable control mRNA which is reasonably abundant in C. albicans mRNA and which remains at a constant level during morphogenesis (2).

Production of C. albicans TBP in vitro.

The 716-nucleotide C. albicans TBP1 open reading frame (ORF) was amplified by PCR, cloned into the pGEN3z-f(+) vector (Promega, Southampton, United Kingdom), and resequenced. TBP1 PCR primers were 5′-CGGGATCCCGCCATGGATTTAAAATTACCCCCA-3′ (BamHI site underlined) and 5′-GCTCTAGAGCTCAATTTTTACGAAATTCATT-3′ (XbaI site underlined) (start and stop codons in italics). The TBP1 ORF was transcribed and translated with a TNT Coupled Reticulocyte Lysate kit, following the manufacturer’s instructions (Promega). Reaction mixtures contained [35S]methionine, and 5-μl aliquots were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (37).

In vitro transcription assays.

In vitro transcription was performed with 20-μl reaction mixtures containing 1 to 2 μl of HeLa cell nuclear extracts (8 U; Promega), 300 ng of cytomegalovirus immediate-early promoter (Promega), 5-μl aliquots of coupled transcription-translation reaction mixtures containing in vitro-synthesized C. albicans TBP (see above), 10 μCi of [α-32P]GTP (3,000 Ci/mmol; Amersham, Little Chalfont, Buckinghamshire, United Kingdom), and 1 μl of RNasin (Promega). Where appropriate, HeLa cell nuclear extracts were heat treated (15 min at 47°C) to inactivate endogenous TBP (32). In vitro transcription reaction mixtures were incubated at 30°C for 60 min, digested with proteinase K (100 μg/ml) for 20 min at 37°C, and subjected to phenol-chloroform extraction, ethanol precipitation, and SDS-PAGE (37).

Gel mobility shift assays.

Gel mobility shift assays were performed (1, 5, 10, 14) with coupled transcription-translation reaction mixtures containing C. albicans TBP (see above) or HeLa cell nuclear extracts (Promega). These were incubated with a TATA-containing double-stranded oligonucleotide that binds human TBP (Promega), 5′-GCAGAGCATATAAGGTGAGGTAGGA-3′ 3′-CGTCTCGTATATTCCACTCCATCCT-5′ with two copies of the C. albicans HYR1 TATA sequence (2), 5′-TAATGATTCTATAATACTCCAAAGTTATAATACTCCAAAGTA-3′ 3′-ATTACTAAGATATTATGAGGTTTCAATATTATGAGGTTTCAT-5′ or with a control oligonucleotide containing two copies of a stress-responsive element (STRE), 5′-TGTATAAACCCCCTTTTCTTGGGGCCCCTTTTCTTGGGGGA-3′ 3′-ACATATTTGGGGGAAAAGAACCCCGGGGAAAAGAACCCCCT-5′ Oligonucleotides were end labelled with [γ-32P]ATP (29). Reaction mixtures contained 2 to 4 μl of transcription-translation reaction mixture or HeLa cell nuclear extract, 1 ng of an end-labelled TATA-containing oligonucleotide, 250 ng of poly(dI-dC) · poly(dI-dC), and binding buffer containing 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, and 50 mM Tris-HCl, pH 7.5. Reactions were performed at room temperature for 20 min and run on nondenaturing polyacrylamide gels.

Nucleotide sequence accession numbers.

The TBP1 genomic and cDNA sequences were submitted to the European Molecular Biology Laboratory with the accession numbers U95549 and U95550, respectively.

RESULTS AND DISCUSSION

Isolation of C. albicans TBP1 sequences.

To isolate C. albicans TBP1 cDNAs, the S. cerevisiae SPT15 gene (encoding TBP [17]) was amplified by PCR and used as a probe to screen a C. albicans cDNA library (47). Four cDNAs were isolated from the 50,000 plaques screened, and these were sequenced to completion. The cDNAs, which were derived from the same locus, displayed strong sequence similarity to the S. cerevisiae SPT15 gene and were named TBP1 (TATA-binding protein 1 gene). A 1.4-kb TBP1 cDNA was then used to isolate the corresponding gene from a C. albicans genomic library. The gene sequence (Fig. 1) was identical to that of the cDNA, indicating that the C. albicans TBP1 gene does not have an intron. The TBP1 sequence has a 716-nucleotide ORF with the potential to encode a 238-amino acid polypeptide with a molecular mass of 27 kDa. A comparison of the predicted amino acid sequences of TBPs from a range of organisms (Fig. 2) reveals strong conservation (over 80% amino acid sequence identity) within their C-terminal domains. However, the N-terminal domains of the fungal, fly, nematode, slime mold, plant, and human TBPs show considerable sequence divergence.

FIG. 1.

FIG. 1

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Nucleotide and predicted amino acid sequences of the C. albicans TBP1 gene. A TATA-like sequence at nucleotide 107 and the initiation codon at 214 are underlined. The single CTG codon in TBP1 at position 892 (underlined) has been decoded as serine and not leucine (39).

FIG. 2.

FIG. 2

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Comparison of the predicted amino acid sequences of TBPs from C. albicans, S. cerevisiae, Homo sapiens, D. discoideum, D. melanogaster, C. elegans, and Arabidopsis thaliana. Accession numbers are as follows: C. albicans, U95549 and U95550;S. cerevisiae, M29459; A. nidulans, U28332; human, M55654; D. discoideum, M64861; D. melanogaster, U11718; C. elegans, L07754; and Arabidopsis thaliana, X54995 and X54996. Asterisks indicate amino acid residues that are identical in all sequences, while dots denote those identical in 6 of the 9 sequences. Residues highlighted in bold in the Arabidopsis thaliana II sequence are known to contact DNA (24). Residues are underlined if they are common to the fungal TBPs but differ from those in the human sequence. S341 is double underlined because it is encoded by a CTG codon (Fig. 1).

A single TBP1 locus in C. albicans.

TBP is encoded by a single locus in S. cerevisiae, Aspergillus nidulans, Drosophila melanogaster, Caenorhabditis elegans, Dictyostelium discoideum, and humans (6, 20, 21, 26, 28), but by two loci in Arabidopsis (15). Southern blots of C. albicans genomic DNA hybridized with the TBP1 cDNA at high stringencies revealed a single band in most lanes (Fig. 3A), suggesting that there is a single TBP1 locus in C. albicans. Three bands were observed with EcoRI, suggesting heterozygosity at an internal EcoRI site not present in the cDNA clone.

FIG. 3.

FIG. 3

FIG. 3

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Southern and Northern blot analyses of C. albicans TBP1. (A) Southern blot of C. albicans 3153A DNA digested with BglII, BamHI, EcoRI, EcoRV, HindIII, or PstI and probed with the TBP1 cDNA. (B) Northern blot of C. albicans 3153A RNA prepared from cells grown at 25 or 37°C in the presence (+) or absence (−) of serum. Cells grown at 37°C with serum were all (>95%) in the hyphal form, whereas cells grown under the other conditions were in the yeast form. The upper panel shows the ethidium bromide-stained gel which was subsequently probed with the TBP1 cDNA (lower panel). TBP1 mRNA levels were quantified by phosphorimaging.

TBP1 expression during morphogenesis.

TBP1 expression during morphogenesis was analyzed by Northern blotting (Fig. 3B). A TBP1 mRNA of about 1.4 kb was observed under all conditions studied, indicating that full-length cDNA clones had been isolated. mRNA levels in control cells growing in the yeast form were compared with those in hyphal cells induced by serum addition and temperature elevation. TBP1 mRNA levels were about 1.5-fold higher in the yeast form than in the hyphal form of C. albicans. The transcriptional activities of numerous C. albicans genes change during morphogenesis (9, 48–51), and therefore small changes in TBP1 expression are not surprising.

C. albicans TBP function.

As described above, the C. albicans TBP1 gene has the potential to encode a protein of 27 kDa. To test this, the TBP1 ORF was cloned into pGEM3z-f(+) and expressed by in vitro transcription and translation (Fig. 4A). SDS-PAGE analysis revealed a 35S-protein with an Mr of about 27,000. Also, recombinant expression of C. albicans TBP in Escherichia coli yielded a protein of an equivalent size (not shown), confirming the predicted size of 27 kDa, which is consistent with the sizes of TBPs from other organisms (6, 15).

FIG. 4.

FIG. 4

FIG. 4

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C. albicans TBP function in vitro. (A) SDS-PAGE analysis of 35S-TBP generated by in vitro transcription and translation of the C. albicans TBP1 ORF (Materials and Methods). Lane 1, control reaction mixture containing no TBP1 cDNA; lane 2, lysate containing TBP1 cDNA showing the 35S-TBP polypeptide with an Mr of 27,000. (B) Gel-shift assays comparing in vitro-synthesized C. albicans TBP (CaTBP) with HeLa cell extracts containing TBP. These were incubated with synthetic double-stranded oligonucleotides carrying a HeLa TATA box, the C. albicans HYR1 TATA box, or a control oligonucleotide lacking TATA (STRE). Competitor was a 10-fold excess of unlabelled HeLa or HYR1 TATA oligonucleotide. Closed arrowhead, complex formation; open arrowheads, uncomplexed oligonucleotides. (C) PAGE analysis of 32P-labelled in vitro transcription products. Transcription from the cytomegalovirus immediate-early promoter was performed with HeLa cell nuclear extracts with (+) or without (−) prior heat treatment to inactivate endogenous TBP. CaTBP, in vitro-synthesized C. albicans TBP; M, pBR322-MspI size markers.

To determine whether C. albicans TBP1 encodes a functional TBP, its ability to bind a TATA box in vitro was tested (Fig. 4B). C. albicans TBP was synthesized by in vitro transcription and translation, and DNA binding was assayed with two TATA-containing oligonucleotides: a commercial oligonucleotide used for human TBP assays, and a second nucleotide carrying the TATA region from the hyphally regulated C. albicans HYR1 gene (2). As expected, HeLa cell extracts formed specific complexes with the first TATA sequence. Significantly, the C. albicans TBP formed a specific complex with the HYR1 TATA element but not with an oligonucleotide containing an STRE, indicating that the TBP1 gene encodes a sequence-specific DNA-binding protein. The data also indicate that, in contrast to S. cerevisiae TBP (17), the C. albicans protein does not require transcription factor IIA to form a DNA-protein complex.

Interestingly, HeLa cell extracts formed only weak complexes with the HYR1 TATA region, and C. albicans TBP did not form a complex with the human TBP oligonucleotide under the conditions studied (Fig. 4B). TBP is known to interact with 8 bp of DNA (24), and hence the differing sequences of the two TATA boxes and/or the fact that the HYR1 TATA region contains two adjacent TATA elements probably influenced their interactions with the HeLa and C. albicans TBP.

The basis for differences in DNA sequence preference must lie in the structural divergence of TBPs from different organisms. A high degree of sequence divergence exists in the N-terminal domain corresponding to residues 1 to 173 of the C. albicans sequence (Fig. 2). However, the sequences both necessary and sufficient for DNA binding and for basal transcription in vitro lie in the C-terminal domain (19, 36) at nucleotides corresponding to residues 174 to 352 of the C. albicans sequence. This domain displays at least 80% amino acid sequence identity among the various organisms. Residues that contact DNA (24) are almost completely conserved among the nine sequences shown, but there are 15 differences between C. albicans and human TBPs which lie very close to residues that contact DNA (Fig. 2). These might account for the different DNA-binding specificities of C. albicans and human TBP.

C. albicans TBP function was analyzed further by testing its ability to support basal transcription in vitro. C. albicans TBP was synthesized by in vitro transcription and translation and added to HeLa nuclear extracts in which the endogenous TBP had been inactivated by heat treatment (32). The C. albicans TBP restored transcriptional activity to these TBP-depleted extracts (Fig. 4C), as does human and S. cerevisiae TBP (21, 22). This confirmed that C. albicans TBP1 encodes a functional TBP. This is the first paper to report the cloning of a functional basal transcription initiation factor from C. albicans.

C. albicans TBP functions in S. cerevisiae.

To determine whether C. albicans TBP functions in S. cerevisiae, we tested whether TBP1 could rescue a Δspt15 null mutation. A heterozygous strain (SPT15/spt15::HIS3 his3/his3) was transformed with the C. albicans TBP1 genomic clone or with the vector alone (YCp50). Transformants were sporulated, and tetrads were dissected (Fig. 5A). As expected, all tetrads derived from transformants harboring vector alone displayed two viable and two nonviable spores, and all viable spores were histidine auxotrophs. In contrast, four viable spores were obtained from transformants harboring the C. albicans TBP1 gene. These tetrads all segregated 2:2 with respect to histidine prototrophy, correlating with the segregation of the spt15::HIS3 and SPT15 alleles. Therefore, C. albicans TBP functions in S. cerevisiae.

FIG. 5.

FIG. 5

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C. albicans TBP functions in S. cerevisiae. (A) Tetrad analysis of S. cerevisiae YAB668 (his3/his3 SPT15/spt15::HIS3) containing the vector YCp50 uncovered two viable histidine-requiring haploids. The same strain containing C. albicans TBP1 on YCp50 (CaTBP) yielded four viable haploids: the two histidine prototrophs grew more slowly than the two histidine auxotrophs. (B) Growth in YPD of haploid S. cerevisiae strains containing C. albicans TBP1 on YCP50: SPT15, squares; spt15::HIS3, circles. Errors from quadruplicate experiments are too small to show up in the figure.

Spt15::HIS3 haploids containing the C. albicans TBP1 gene formed relatively small colonies compared to those formed by wild-type SPT15 strains (Fig. 5A), and the relatively slow growth of the mutants was confirmed in liquid culture (Fig. 5B), suggesting that C. albicans TBP might not fully complement the S. cerevisiae spt15 mutation. Hence, the behavior of spt15::HIS3 strains containing the C. albicans TBP1 gene was analyzed further. Growth was tested on YPD over a range of temperatures (25 to 35°C), on different carbon sources (glucose, galactose, or glycerol), in the presence of 50 mM aminotriazole (which induces an amino acid starvation response), and in the presence of 0.25 mM Cu2+. Spt15::HIS3 cells containing the C. albicans TBP1 gene were viable under all conditions analyzed but consistently grew with a doubling time that was about 80% of that of SPT15 cells (data not shown). This indicates that C. albicans TBP can support transcriptional activation by Gal4p, Gcn4p, and Ace1p in S. cerevisiae, in contrast to the A. nidulans TBP, which only partially complements an spt15 mutation in S. cerevisiae (26).

Fungus-specific TBP residues.

Given the rising incidence of life-threatening systemic fungal infections, there is a need to identify novel antifungal targets. Proteins such as TBP that perform essential functions provide attractive targets, but antifungal therapies would have to focus on critical differences between the mammalian and fungal proteins. Interestingly, the essential C-terminal domain contains 17 residues that are conserved in fungal TBPs but which differ from human TBP (Fig. 2). Nine of these residues (T178, A184, V235, D242, D243, K245, S248, I324, and E333) lie in, or very close to, residues that contact DNA. The cloning of the TBP1 gene from C. albicans will help us to test the validity of TBP as an antifungal target and to dissect transcriptional regulation in C. albicans in response to specific environmental, developmental, and pathogenic signals.

ACKNOWLEDGMENTS

We thank Mark Payton for the C. albicans genomic library.

P.L. is supported by a grant from the Biotechnology and Biological Sciences Research Council (CEL04563). P.C. is supported by a Wellcome Trust Equipment grant (M/95/2409).

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