UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner.
UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes. UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors. This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster. In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein. At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149). The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone. DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence.
Anderson
S F
SF
Department of Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Steber
C M
CM
Esposito
R E
RE
Coleman
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000032:analysis; D018983:DNA Footprinting; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003850:Deoxyribonuclease I; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008540:Meiosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000032:analysis / Q000494:pharmacology*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C089979:UME6 protein, S cerevisiae; D003850:Deoxyribonuclease I; D015032:Zinc; D003545:Cysteine