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 progression through binding to upstream regions of IME1 and CLN2 in Saccharomyces cerevisiae. Rme1p has three zinc finger segments, and two of them are atypical. To determine DNA binding domain of Rme1p, a series of Rme1p derivatives fused with maltose-binding protein were purified and characterized by gel mobility shift assay. We show that not only three zinc fingers, but also the neighboring C-terminal region is essential for DNA binding. Mutational analysis of this region revealed that basic residues Arg-287, Lys-290, and 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 proline 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-terminal segment is essential and acts as a novel accessory domain for DNA binding by zinc fingers.
Shimizu
M
M
Department of Chemistry, Meisei University, 2-1-1 Hodokubo, Hino, Tokyo, 191-8506 Japan. shimizum@chem.meisei-u.ac.jp
Murase
A
A
Hara
M
M
Shindo
H
H
Mitchell
A P
AP
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C071594:RME1 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA