DNA-binding properties of the yeast transcriptional activator,Gcr1p

In Saccharomyces cerevisiae the GCR1 gene product is required for high-level expression of genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcr1p. The DNA-binding domain of Gcr1p binds DNA with high affinity. The apparent dissociation constant of the Gcr1p DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2·9×10⁻¹⁰ M. However, competition experiments showed that Gcr1p binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA of random sequence to inhibit the formation of nucleoprotein complexes between Gcr1p and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcr1p-binding sites are varied relative to the ends, indicated that Gcr1p–DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.     

Role of the N-terminal region of Rap1p in the transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

In the yeast two-hybrid system, the N-terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two-hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N-terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N-terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N-terminal region of Rap1p. These results suggest that one function of the N-terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein-protein interaction.     

Isolation and sequence of the GCR3 homologue from Candida albicans by complementation of (delta)gcr3 strain of Saccharomyces cerevisiae

To study the function of GCR3, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Candida albicans gene which complements the growth defect of the (delta)gcr3 mutant was isolated. Transformants of this gene also recovered the glycolytic enzyme activities. Its DNA sequencing predicted an 886 amino acid protein with 30.4% identity to the Gcr3p of S. cerevisiae.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation of GCR1, a major transcription factor of glycolytic genes in Saccharomyces cerevisiae, from Kluyveromyces lactis

To study the function of GCR1, a gene involved in the expression of glycolytic genes in Saccharomyces cerevisiae, a Kluyveromyces lactis gene that complements the growth defect of a S. cerevisiae Deltagcr1 mutant was isolated. Introduction of this gene into the Deltagcr1 mutant also restored the activities of glycolytic enzymes. DNA sequencing of KlGCR1 predicted an open reading frame of a 767 amino acid protein with an overall identity of 33% and similarity of 48% to Gcr1p from S. cerevisiae. Its DDBJ/EMBL/GenBank Accession No. is AB046391.