Eukaryotic transcription factors are typically bipartite in nature, with a region (the DNA binding or DB domain) specifically designed to interact with a defined DNA sequence and a region (the transcriptional activation or TA domain) required to interface the factor with the transcriptional machinery. There are several classes of each of these modules, and they are connected together in a variety of ways. Within the transcriptional activation modules, there are domains rich in acidic or basic residues or rich in glutamine, threonine/serine, or isoleucine residues (2
). In this study we defined the transcription activation domains in a pair of C. albicans
transcription factors that share sequence similarity with their S. cerevisiae
homologs only within their DNA binding domains. The Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA binding domain is one of the largest classes of fungal DNA binding proteins, the best characterized of which are Gal4p, Ppr1p, Leu3p, Hap1p, and Put3p. Although the DNA binding sequence of ScPut3p (CGGN10
CCG) is very similar to that of ScGal4p (CGGN11
CCG), the distinction in recognition sequences is conserved; C. albicans
possesses homologs of all of these S. cerevisiae
Gal4-like Zn(II)2Cys6 proteins, including Put3p.
In S. cerevisiae
it was shown that Gal4AD and Gcn4AD have an acidic amino acid-rich nature and are located in the C′ and N′ termini, respectively (13
). We analyzed the transcription activation domains of the C. albicans
) and Gal4p (CaGAL4
) homologs and found that just as in S. cerevisiae
, they are positioned at the N′ and C′ termini of the respective proteins. However, the C. albicans
Gcn4p and Gal4p activation domains do not share sequence similarity either to each other or to the activation domains of their S. cerevisiae
homologs, and C. albicans
Gal4p and Gcn4p have nucleophilic activation domains. Nucleophilic transcriptional activation regions have been previously seen almost exclusively in higher eukaryotic transcription factors (9
). A screen for C. albicans
transcriptional activation domains using a genomic library fused downstream of lexA
yielded an active fragment containing a normally noncoding region that expressed 33% serines and threonines in the fusion construct (data not shown), which also suggests that nucleophilicity can be an important feature of C. albicans
activation domains. The serine and threonine amino acids could potentially be converted into an acidic form by phosphorylation.
The S. cerevisiae
Gal4p and Gcn4p proteins each contain two transcriptional activation domains (13
). In contrast, the C. albicans
Gcn4p and Gal4p homologs appear to each contain only one transcriptional activation domain (Fig. and ). Each of the two ScGcn4p activation domains seems to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of the Gcn4p-dependent genes (13
). These results are consistent with our observation that the C-to-N-terminal deletions within the CaGcn4p activation domain lead to a gradual, rather than to an abrupt, reduction of the transcription-activating abilities of the fusion protein (Fig. ).
To determine when the changes in the activation domains of Gcn4p and Gal4p occurred during the evolution of the yeast species, we used available genomic data of the ascomycota (Schizosaccharomyces pombe, Neurospora crassa, Aspergillus niger, S. cerevisiae, Saccharomyces paradoxus, Saccharomyces mikatae, Saccharomyces kudriavzevii, Saccharomyces bayanus, Candida glabrata, Saccharomyces castellii, Kluyveromyces lactis, Ashbya gossypii, Debaryomyces hansenii, Candida tropicalis, Candida dubliniensis, and C. albicans) (Fig. ). Archiascomycetes were observed to lack the activation domains of either ScGcn4p or CaGcn4p. Euascomycetes possessed the ScGcn4p activation domain II (ADII). We noted that the common ancestor of D. hansenii, C. tropicalis, C. dubliniensis, and C. albicans lost activation domain I (ADI) of ScGcn4p and acquired the activation domain of CaGcn4p. We also observed that the ancestor of C. tropicalis, C. dubliniensis, and C. albicans lacked ScGcn4p ADII (Fig. ). In addition, the ancestor of C. tropicalis, C. dubliniensis, and C. albicans lacked ScGal4p ADI, while the ancestor of D. hansenii, C. tropicalis, C. dubliniensis, and C. albicans lacked ScGal4p ADII (Fig. ). In both cases, D. hansenii represents an intermediate with both S. cerevisiae and C. albicans activation domains. These observations show that the changes in the activation domains of Gal4p and Gcn4p of C. albicans occurred relatively recently on the evolutionary scale.
FIG. 5. Phylogenetic comparison of Gcn4p and Gal4p orthologs across the Ascomycota. The changes in the Gcn4p activation domain of C. albicans occurred relatively recently on the evolutionary scale. We used available genomic data of the Ascomycota Schizosaccharomyces (more ...)
The lack of homology in the activation domains of transcriptional activators between S. cerevisiae and C. albicans might suggest a concomitant reduced structural similarity in the activation domain-interacting complexes between the two species. A pairwise sequence comparison of the transcriptional machinery between C. albicans and S. cerevisiae shows a high level of conservation in the RNA polymerase II complex. The exceptions for this are transcription factor IIA and the Mediator complex: S. cerevisiae and C. albicans show low levels of homology with respect to the proteins of those two complexes, and these are the complexes that interact with transcriptional regulators.
The characterization of the bipartite structure of eukaryotic transcription factors like S. cerevisiae
Gal4p was a fundamental conceptual advance (33
) and has led to important technical developments like the yeast two-hybrid system (17
). In general, C. albicans
transcription factors follow the pattern of distinct DNA binding and transcriptional activation domains, and many show strong sequence similarity, extending to both domains, to specific S. cerevisiae
transcription factors. However, a large number of C. albicans
proteins have strong sequence similarity that is limited only to the DNA binding module of an S. cerevisiae
transcription regulator. We have shown that although the well-studied Gal4p and Gcn4p proteins of S. cerevisiae
share similarity only to the DNA binding regions of the Gcn4p and Gal4p proteins of C. albicans
, the Candida
proteins still contain transcriptional activation capacity. Further work will be necessary to establish the molecular logic of linking common DNA binding modules to distinct activation domains in these two fungi, in particular in cases such as Gcn4p where similar cellular processes are regulated by the two proteins.