Rox1 is a repressor of the hypoxic genes of Saccharomyces cerevisiae. It binds to a specific hypoxic consensus sequence in the upstream region of these genes and represses transcription in conjunction with the general repression complex Tup1-Ssn6. In this study, we demonstrated that the first 100 amino acids comprising the HMG domain of Rox1 were responsible for DNA binding and that when bound, Rox1 bent DNA at an angle of 90 degrees. A mutational analysis resulted in the isolation of seven missense mutations, all located within the HMG domain, that caused loss of DNA binding. The effect of these mutations on the structure of Rox1 was evaluated on the basis of the homology between Rox1 and the human male sex-determining protein SRY, for which a structural model is available. The failure to isolate missense mutations in the carboxy-terminal three-quarters of the protein prompted a deletion analysis of this region. The results suggested that this region was responsible for the repression function of Rox1 and that the repression information was redundant. This hypothesis was confirmed by using a set of fusions between sequences encoding the GAL4 DNA-binding domain and portions of ROX1. Those fusions containing either the entire carboxy-terminal region or either half of it were capable of repression. Repression by selected fusions was demonstrated to be dependent on Ssn6.

Conjugative F-plasmid transfer is mediated by the transfer (tra) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli. TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene. The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY. Using primer-extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription. traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein. Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene.