Rap1p's function at glycolytic enzyme gene UAS elements is to facilitate
binding of Gcr1p at adjacent sites. There are two mechanistic models for
Rap1p's action. One model proposes that Rap1p-induced DNA-bending alters
the topology of adjacent Gcr1p-binding sites thereby creating high-affinity sites. The second model proposes that interactions between
Rap1p and Gcr1p stabilize Gcr1p on its binding site. Rap1p has a modular
domain structure. In its amino-terminus there is an asymmetric DNA-bending domain that is distinct from its DNA-binding domain which
resides in the middle of the protein. In the carboxyl-terminus lie the
silencing and putative activation domains of Rap1p. We carried out a
molecular dissection of Rap1p to identify domains contributing to its
ability to facilitate binding 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 DNA-binding domain of Rap1p,
while competent to bind DNA, was unable to facilitate binding of Gcr1p.
Full-length and the amino- and carboxyl-truncations of Rap1p were able
to facilitate binding of Gcr1p at an appropriately spaced binding site.
When spacing between Rap1p- and Gcr1p-bindings sites were altered by
insertion of five nucleotides, the ability to form ternary complexes
between Rap1p*DNA*Gcr1p was inhibited by all but the DNA-binding of
Rap1p itself; however, the ability of each individual protein to bind
the DNA probe was unaffected.
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