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.     

trp repressor interactions with the trp aroH and trpR operators. Comparison of repressor binding in vitro and repression in vivo

Interaction of the Escherichia coli trp repressor with the promoter-operator regions of the trp, aroH and trpR operons was studied in vivo and in vitro. The three operators have similar, but non-identical, sequences; each operator is located in a different segment of its respective promoter. In vivo repression of the three operons was measured using single-copy gene fusions to lacZ. The extent of repression varied from 300-fold for the trp operon, to sixfold for the aroH operon and threefold for the trpR operon. To determine whether differential binding of repressor to the three operators was responsible for the differences in repression observed in vivo, three in vitro binding assays were employed. Restriction-site protection, gel retardation and DNase footprinting analyses revealed that repressor binds to the three operators with almost equal affinity. It was also shown in an in vivo competition assay that repressor binds approximately equally well to each of the three operators. It is proposed that the differential regulation observed in vivo may be due to the different relative locations of the three operators within their respective promoters.     

Mutational analysis of the operators of bacteriophage lambda

Summary O ^c mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at O _R1 and O _R2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promoter sites.     

Probing co-operative DNA-binding in vivo. The lac O1:O3 interaction

The lac primary (O1) and weak upstream pseudo (O3) operators contained on a plasmid were footprinted in vivo in order to determine whether they act co-operatively in binding lac repressor in the cell. The occupancy at O3 by lac repressor was substantially reduced upon deletion of the lac primary operator, demonstrating co-operativity at a distance. Plots of operator occupancy versus active repressor concentration were obtained for each operator by treating the cells with different amounts of the lac inducer isopropyl-beta-D-thiogalactoside and probing lac repressor binding. This analysis can be used to obtain relative binding constants in vivo and demonstrates that O3 binds repressor only 10.3-fold less tightly than O1 in their co-operative interaction. The removal of DNA torsional tension in vivo by the use of coumermycin leads to the same loss of binding at O3 as does deleting O1. These in-vivo results are analogous to the in-vitro situation, where O3 binds repressor strongly in a DNA repression loop only on supercoiled templates.     

Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter.

Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter. They were tested in vivo for function in the presence and absence of lac repressor. We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene. When lac operators are inserted at both sites, we found a greater than 150-fold repression. The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators.