UME6, a negative regulator of meiosis insaccharomyces cerevisiae, contains a C-terminal Zn₂Cys₆binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner

Abstract

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 "7'c-x~-c-x6-c-x6-C-X,-C-X,-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cysrich 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 overproduced within the last 11 1 amino acid residues of UME6, UME6(11 I), forms a binuclear cluster and exhibits a Zn-dependent binding to the URSl DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 k 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 k 0.1 mol Cd/mol protein.At 5 "C, 1'3Cd2UME6(l 11)shows two Il3Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for "3CdzGAL4(149). The magnitude of these chemical shifts suggests that each 'I3Cd nucleus is coordinated to four -S-ligands, compatible with a II3Cdz cluster structure in which two thiolates form bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URSl sequence than the zinc binuclear cluster domain alone. DNase 1 footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URSl sequence.

Analysis of a Meiosis-Specific URS1 Site: Sequence Requirements and Involvement of Replication Protein A

“…This prediction is supported by the observation that the CAR1, SPO13, and IME2 URS1 sites require the presence of Ume6, also known as Car80, to repress transcription (6,34,42). Ume6 contains a Cys 6 cluster, as does the DNA-binding domain of the transcriptional activator Gal4, and purified Ume6 protein has been shown to directly bind to the SPO13 URS1 (URS1 SPO13 ) site (3,42). The Ume6 protein is also required for the URS1 site to function as a meiosisspecific activator site (6,40,42).…”

“…Free probes are shown in lanes 1, 6, and 11. Seventy nanograms of purified RPA was used in lanes 9 and 10; 200 ng of purified RPA was used in lanes 4, 5, 14, and 15; 30 g of mitotic cells extracts (LNY3) was used in lanes 2,3,7,8,12, and 13; 5 l of 1:100-fold-diluted RPA antibody (Ab) (against the 69-kDa subunit) was added to the reaction mixture before the addition of DNA in lanes 3,5,8,10,13 These small differences may be important distinguishing a meiotic URS1 site from a nonmeiotic site.…”

“…Multiple DNA-binding activities have been reported for binding to various URS1 sites (3,25,27,34,42,47). The trimeric RPA has been purified as the URS1 C -binding factor (27,28).…”

“…The trimeric RPA has been purified as the URS1 C -binding factor (27,28). Moreover, it was shown that the Ume6 (Car80) protein mediates transcriptional repression through URS1 C (34) and that it binds to the URS1 site in SPO13 (3,42). DNAbinding activities in EMSAs have been reported for the INO URS1 and TRK2 URS1 sites, called URSBf and URSF, respectively (25,47).…”

The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast

“…The UME6 gene in Saccharomyces cerevisiae is a major regulator mediating some of these responses. UME6 encodes a DNA-binding protein (1,2) that associates with the upstream regulatory sequence URS1 A (consensus 5Ј-TCGGCGGCT-3Ј) to regulate transcription of genes responding to metabolites such as glucose, nitrogen and inositol (3)(4)(5)(6)(7)(8) and, in some cases, with a noncanonical URS1 (URS1 B ; 5Ј-SGWGGMRRNANW-3Ј) to regulate genes involved in DNA repair (9). Although Ume6 is dispensable for growth, it controls efficient mating of haploids and is essential for the initiation and progression of meiosis (2,10).…”