Identification and characterisation of two transcriptional repressor elements within the coding sequence of the Saccharomyces cerevisiae HXK2 gene.
A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different HXK2::lacZ gene fusion integrated at the URA3 locus. These HXK2::lacZ fusions differ in the amount of the HXK2 gene (encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the coding region between +39 and +404 bp is involved in repressing gene expression in a carbon source dependent manner. A series of deletions of this HXK2 coding region were constructed and fused upstream of a minimal CYC1::lacZ promoter. beta-Galactosidase activities on glucose or ethanol growth yeast calls revealed that two different regulatory elements are present in this DNA region. Gel mobility shift analysis and in vitro DNase I footprinting have shown that proteins bind specifically to two downstream repressor sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and +251) that influence the rate of HXK2 transcription when ethanol is used as carbon source by Saccharomyces cerevisiae. We identified and partially purified a 18 kDa protein that binds specifically to synthetic double-stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence. Our data suggest that p18 synthesis is under the control of genes involved in glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1).
Herrero
P
P
Instituto Universitario de Biotecnología de Asturias (IUBA), Departamento de Bioquímica y Biologia Molecular, Universidad de Oviedo, Spain.
Ramírez
M
M
Martínez-Campa
C
C
Moreno
F
F
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D006593:Hexokinase; D001616:beta-Galactosidase; D005947:Glucose