Approaches to the study of Rox1 repression of the hypoxic genes in the yeast Saccharomyces cerevisiae

Abstract

The yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen tension by changing patterns of gene expression. One set of genes that responds to this environmental cue is the hypoxic genes. Oxygen levels are sensed by changes in heme biosynthesis, which controls the transcription of the ROX1 gene, encoding a protein that binds to the regulatory region of each hypoxic gene to repress transcription. Several experimental molecular and genetic approaches are described here to study Rox1 repression. Derepression of the hypoxic genes is rapid, and one model for such a response requires that Rox1 have a short half-life. This was demonstrated to be the case by immunoblotting using a c-myc epitope-tagged protein. Rox1 repression is mediated through the general repressors Ssn6 and Tup1. To explore possible interactions among these proteins, all three were expressed and partially purified using a baculovirus expression system and histidine-tagged proteins. The effect of Ssn6 and Tup1 on the formation of Rox1-DNA complexes was explored using these purified proteins by both electrophoretic mobility shift and DNase I protection assays. We found that Rox1 DNA-binding activity decayed rapidly and that Ssn6 could stabilize and restore lost activity. Finally, genetic selections are described for the isolation of loss-of-function mutations in Rox1. Also, schemes are proposed for the reversion of such mutations. These selections have been extended to genetic analyses of the TUP1 and SSN6 genes.