The Wrapping Loop and RCT Domain of Yeast Rap1 Modulate Access to Different DNA Binding Modes [Molecular Biophysics]

March 24th, 2015 by Feldmann, E. A., De Bona, P., Galletto, R.

Budding yeast Rap1 is a specific double-stranded DNA-binding protein involved in repression and activation of gene transcription and in the establishment of the nucleoprotein complex formed at telomeres. The DNA-binding domain (DBD) of Rap1 forms a high-affinity complex with DNA where both Myb-like domains bind to the recognition site. However, we recently showed that the DBD can also access an alternative, lower affinity DNA-binding mode where a single Myb-like domain binds. This results in Rap1-DNA complexes with stoichiometry higher than previously anticipated. In this work we show that the ability of the DBD to form higher stroichiometry complexes on DNA is maintained also in larger Rap1 constructs. This indicates that transition between at least two DNA-binding modes is a general property of the protein and not a specific feature of the DBD in isolation. The transition between binding modes is modulated by the C-terminal wrapping loop within the DBD, consistent with the proposed model that the transient opening of this region allows a switch between binding modes. Finally, we provide evidence that the Rap1 C-terminus domain (RCT) interacts with the DNA-binding domain, suggesting a complex network of interactions that couples changes in conformation of the protein to the binding of its DNA recognition sequence.