MUTATIONAL ANALYSIS OF ROX1, A DNA-BENDING, REPRESSOR OF HYPOXIC GENES IN SACCHAROMYCES-CEREVISIAE

Rox1 is a repressor of the hypoxic genes of Saccharomyces cerevisiae. It binds to a specific hypoxic consensus sequence in the upstream regi on of these genes and represses transcription in conjunction with the general repression complex Tup1-Ssn6. In this study, we demonstrated t hat the first 100 amino acids comprising the HMG domain of Rox1 were r esponsible for DNA binding and that when bound, Rox1 bent DNA at an an gle of 90 degrees. A mutational analysis resulted in the isolation of seven missense mutations, all located within the RR?IG domain, that ca used loss of DNA binding. The effect of these mutations on the structu re of Rox1 was evaluated on the basis of the homology between Rox1 and the human male sex-determining protein SRY, for which a structural mo del is available. The failure to isolate missense mutations in the car boxy-terminal three-quarters of the protein prompted a deletion analys is of this region. The results suggested that this region was responsi ble for the repression function of Rox1 and that the repression inform ation was redundant. This hypothesis was confirmed by using a set of f usions between sequences encoding the GAL DNA-binding domain and porti ons of ROX1. Those fusions containing either the entire carboxy-termin al region of either half of it were capable of repression. Repression by selected fusions was demonstrated to be dependent on Ssn6.