In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.Dimerization is a common mechanism by which the activity of numerous important biological macromolecules can be regulated. These molecules include receptors for growth hormones (1, 2), steroid hormones (3), other cellular signals (4, 5), and numerous transcription factors (6, 7). The transcriptional activators of the yeast Gal4 family also require dimerization for DNA binding and transcriptional activation (8,9). This family includes at least 52 transcription factors that control a wide array of diverse processes ranging from carbon source utilization to oxygen utilization and drug resistance (8, 9). These members all contain a C6 zinc cluster that recognizes a CGG triplet (9 -15). Although the DNA binding properties of the C6 zinc cluster proteins are well characterized, the molecular mechanisms by which these members act to control transcription in response to various signals are largely unclear. Interestingly, recent data suggest that, like steroid hormone receptors, certain members of the yeast Gal4 family such as Hap1 and Pdr1 (16,17) are regulated by Hsp90 and Hsp70 molecular chaperones. In particular, Hap1 is a heme-responsive transcriptional activator, which promotes transcription of genes required for respiration and for controlling oxidative damage in response to oxygen/heme (18 -20). In the absence of heme, Hap1 is bound to cellular proteins including Hsp82 (the yeast homologue of Hsp90) and Ydj1, forming a higher order complex termed HMC 1 (17,21,22). Hap1 DNA binding and transcriptional activities are repressed in this complex. Heme disrupts the HMC and permits Hap1 to bind to DNA as a dimer with high affinity, thereby activating transcription. The formation and disruption of the HMC are the key events in Hap1 repression in the absence of heme and subsequent activation by heme. ...

Mitochondrial DNA from Triticum timopheevi has a chimeric gene, orf256, upstream of coxI. This gene is cotranscribed with coxI in cytoplasmic male sterile plants and produces a 7-kDa protein which is not produced in fertile or fertility-restored plants. T. aestivum, the nuclear donor in sterile plants, does not have orf256. Analysis by polymerase chain reaction of DNA from barley, rye, Aegilops bicornis, Ae. searsii, Ae. sharonensis, Ae. speltoides, Ae. tauschii, T. monococcum, and T. turgidum was done with oligonucleotide primers designed to detect orf256 or coxI sequences. Except for T. turgidum, these plants have various elements of the orf256 sequence over a 1-kb length of DNA immediately upstream of coxI in exactly the same arrangement as is found in the coxI region of T. timopheevi. Only T. timopheevi and Ae. speltoides have orf256 transcripts, and only cytoplasmic male-sterile plants involving these two species as maternal donors produce a protein from orf256. Part of an orf256-like sequence is present in T. turgidum but is at least slightly different in arrangement relative to coxI, as compared with the sequence in T. timopheevi. Neither maize nor sorghum have the orf256 sequence.