A C-terminal segment with properties of alpha-helix is essential for DNA binding and in vivo function of zinc finger protein Rme1p

Rme1p plays important roles in the control of meiosis and in cell cycle pro gression through binding to upstream regions of IME1 and CLN2 in Saccharomy ces cerevisiae. Rme1p has three zinc finger segments, and two of them are a typical. To determine DNA binding domain of Rme1p, a series of Rme1p deriva tives fused with maltose-binding protein were purified and characterized by gel mobility shift assay. We show that not only three zinc fingers, but al so the neighboring C-terminal region is essential for DNA binding. Mutation al analysis of this region revealed that basic residues Arg-287, Lys-290, a nd Arg-291 and the hydrophobic residues Phe-288, Leu-292, Ile-295, and Leu- 296 are critical for DNA binding. In addition, double substitutions by prol ine at Asn-289 and Lys-293, each of which was not essential for DNA binding , abolished DNA binding. These results suggest that the C-terminal segment forms an amphipathic helical structure. Furthermore, it was shown that the mutations in the important basic residues abolish or impair Rme1p function in vivo for repression and inhibition of spore formation. Thus, the C-termi nal segment is essential and acts as a novel accessory domain for DNA bindi ng by zinc fingers.