Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

Abstract

GCRI gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae.Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTpI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcrl deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcrl mutant strain. These data demonstrate that the UAS of TPI contains two GCRl-binding sites which are occupied in vivo. Furthermore, adjacent RAPl/GRFI/TUF-and REB1/GRF2/QBP/Y-binding sites in UASTp, were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO], PYK, and ADHi, all of which are dependent on GCRI gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence

Phylogenetic footprinting reveals multiple regulatory elements involved in control of the meiotic recombination gene, REC102

“…In mitotic cells Gcr1p acts as a positive activator of the glycolytic genes, which normally requires adjacent binding of Rap1p; a single Gcr1p binding site does not itself have UAS activity (e.g. Stanway et al, 1989;Huie et al, 1992;Lopez et al, 1998). However, the Rap1p binding sites in the REC102 promoter are not adjacent.…”

Cryptons: a group of tyrosine-recombinase-encoding DNA transposons from pathogenic fungi

“…The matching proteins include Saccharomyces cerevisiae Msn1p (multi-copy suppressor of SNF1; Estruch & Carlson, 1990), Hot1p (high osmolarity-induced transcription factor; Rep et al, 1999), and Gcr1p (transcriptional regulator of glycolytic genes; Holland et al, 1987) and the Kluyveromyces lactis homologue (KlGcr1p; Haw et al, 2001). In the case of Gcr1p, the region that matches the crypton proteins has been shown to bind DNA with high affinity (Huie et al, 1992;Huie & Baker, 1996). Msn1p is also known to be a DNA-binding protein (Estruch & Carlson, 1990).…”

Molecular genetics of 6‐phosphofructokinase in Pichia pastoris

“…A putative ADR1-binding site (TCTCC) involved in glucose-repression (Denis and Young, 1983) and a consensus GCR1-binding site (CA-TCC) could be identified at −80 bp and −707 bp, respectively. The latter promoter element was found in front of many genes encoding glycolytic enzymes and a GCR1 gene function is known to be required for high-level glycolytic gene expression in S. cerevisiae (Huie et al, 1992;Uemura and Jigami, 1995).…”