The DNA binding protein Rfg1 is a repressor of filamentation in Candida albicans.

We have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor.

Characterization of the DNA binding and bending HMG domain of the yeast hypoxic repressor Rox1.

The yeast Rox1 hypoxic transcriptional repressor protein binds to and bends a specific DNA sequence through an HMG domain located at the N-terminus. To better understand the structure of Rox1 and how it interacts with DNA, 38 missense mutations in the HMG domain were isolated through a combination of random and site-directed mutageneses, the latter directed to two Ile residues that play an important role in DNA recognition and bending by HMG domains. The mutants were characterized in terms of their ability to repress the hypoxic gene ANB1 and the auto-repressed ROX1 gene in vivo. The mutant HMG domains were fused to maltose binding protein and expressed in and purified from Escherichia coli and their relative affinities for DNA and ability to bend DNA were determined. A model of the structure of the Rox1 HMG domain was derived using sequence similarities between Rox1 and the human protein SRY, the structure of which has been determined. The results of the mutational analysis are interpreted in terms of the model structure of Rox1.