Specificity and Regulation of DNA Binding by the Yeast Glucose Transporter Gene Repressor Rgt1

Abstract

Rgt1 is a glucose-responsive transcription factor that binds to the promoters of several HXT genes encoding glucose transporters in Saccharomyces cerevisiae and regulates their expression in response to glucose. Rgt1 contains a Zn 2 Cys 6 binuclear cluster responsible for DNA binding. Most proteins that contain this sequence motif bind as dimers to regularly spaced pairs of the sequence CGG. However, there are no CGG pairs with regular spacing in promoters of genes regulated by Rgt1, suggesting that Rgt1 binds as a monomer to CGG or to another sequence. We identified the Rgt1 consensus binding site sequence 5-CGGANNA-3, multiple copies of which are present in all HXT promoters regulated by Rgt1. Rgt1 binds in vivo to multiple sites in the HXT3 promoter in a nonadditive, synergistic manner, leading to synergistic repression of HXT3 transcription. We show that glucose inhibits the DNA-binding ability of Rgt1, thereby relieving repression of HXT gene expression. This regulation of Rgt1 DNA-binding activity is caused by its glucose-induced phosphorylation: the hyperphosphorylated Rgt1 present in cells growing on high levels of glucose does not bind DNA in vivo or in vitro; dephosphorylation of this form of Rgt1 in vitro restores its DNA-binding ability. Furthermore, an altered Rgt1 that functions as a constitutive repressor remains hypophosphorylated when glucose is added to cells and binds DNA under these conditions. These results suggest that glucose regulates the DNA-binding ability of Rgt1 by inducing its phosphorylation.

Connection Between the Rag4 Glucose Sensor and the KlRgt1 Repressor in Kluyveromyces lactis

“…In the absence of glucose, ScRgt1 from S. cerevisiae binds to the HXT gene promoters and represses their transcription (Ozcan et al 1996;Flick et al 2003;Kim et al 2003;Mosley et al 2003;Polish et al 2005). Some reports also suggest that in the presence of glucose, ScRgt1 behaves as a transcriptional activator of the HXT genes (Ozcan and Johnston 1995;Ozcan et al 1996;Polish et al 2005).…”

“…Chromatin immunoprecipitation : Chromatin immunoprecipitation (ChIP) experiments were carried out as previously reported (Kim et al 2003) using 10 10 cells cross-linked with formaldehyde (final concentration of 1%) by incubation for 15 min at room temperature. The reaction was quenched with glycine (final concentration of 125 mm).…”

Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I

“…The glucose signal induces this expression by influencing the function of the Rgt1 transcriptional repressor. In the absence of glucose, Rgt1 is functional and binds to promoters of HXT genes and represses their expression (2,3). Glucose derepresses HXT gene expression by inhibiting Rgt1 repressor function.…”

Isolation and characterization of theLGT1gene encoding a low-affinity glucose transporter fromTorulaspora delbrueckii

“…Cells were grown on SD-maltose, washed with chilled water, and transferred (OD 640 = 0.2-0.3) to SD medium containing 2% glucose. Kim et al, 2003). We found three sequences, −410/−415, −421/−416 and −680/−685 containing a perfect Mig1p-binding GC box, (G/C)(C/T)GGGG (Nehlin and Ronne, 1990).…”