MULTIPLE DOMAINS OF REPRESSOR ACTIVATOR PROTEIN-1 CONTRIBUTE TO FACILITATED BINDING OF GLYCOLYSIS REGULATORY PROTEIN-1

The function of repressor activator protein 1 (Rap1p) at glycolytic en zyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and p utative activation domains. We carried out a molecular dissection of R ap1p to identify domains contributing to its ability to facilitate bin ding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p . DNA . Gcr1p depended on the presence of binding sites for both prote ins in the probe DNA. The DNA-binding domain of Rap1p, although compet ent to bind DNA was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were ea ch able to facilitate binding of Gcr1p at an appropriately spaced bind ing site. Under these conditions, Gcr1p displayed an approximately 4-f old greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was alte red by insertion of five nucleotides, the ability to form ternary Rap1 p . DNA . Gcr1p complexes was inhibited by all but the DNA-binding dom ain of Rap1p itself; however, the ability of each individual protein t o bind the DNA probe was unaffected.