Specialized Rap1p/Gcr1p transcriptional activation through Gcr1p DNA contacts requires Gcr2p, as does hyperphosphorylation of Gcr1p.

The multifunctional regulatory factor Rap1p of Saccharomyces cerevisiae accomplishes one of its tasks, transcriptional activation, by complexing with Gcr1p. An unusual feature of this heteromeric complex is its apparent capacity to contact simultaneously two adjacent DNA elements (UASRPG and the CT box, bound specifically by Rap1p and Gcr1p, respectively). The complex can activate transcription through isolated UASRPG but not CT elements. In promoters that contain both DNA signals its activity is enhanced, provided the helical spacing between the two elements is appropriate; this suggests that at least transient DNA loop formation is involved. We show here that this CT box-dependent augmentation of Rap1p/Gcr1p activation requires the presence of a third protein Gcr2p; the Gcr2- growth defect appears to result from a genome-wide loss of the CT box effect. Interestingly, a hyperphosphorylated form of Gcr1p disappears in delta gcr2 cells but reappears if they harbor a doubly point-mutated GCR1 allele that bypasses the Gcr2- growth defect. Gcr2p therefore appears to induce a conformation change in Gcr1p and/or stimulate its hyperphosphorylation; one or both of these effects can be mimicked in the absence of GCR2 by mutation of GCR1. This improved view of Rap1p/Gcr1p/Gcr2p function reveals a new aspect of eukaryotic gene regulation: modification of an upstream activator, accompanied by at least transient DNA loop formation, mediates its improved capacity to activate transcription.