Two alternative pathways of transcription initiation in the yeast negative regulatory gene GAL80.
The yeast GAL80 gene, encoding a negative regulatory protein of galactose-inducible genes, shows both constitutive and galactose-inducible expression. The inducible transcription is under the control of Gal4p, a common activator for the galactose-inducible genes, which binds to an upstream activation sequence, called UASG, spanning between -105 and -89 in the 5'-flanking region of GAL80. Here we demonstrate that the constitutive transcription started at +1, whereas the inducible transcription occurs from a set of downstream sites at +37, +47, +56, and +67. Both transcriptions were enhanced 10-fold by another UAS, whose 5' boundary is located between -195 and -185. Gal4p stimulated transcription, which depends on the TATA box located at -20, from all the downstream sites. By contrast, the constitutive transcription depended on a small region of less than 16 bp long encompassing the +1 site, which directed transcription even in the absence of both the TATA box and the UASs. When a fragment covering that region was inserted immediately upstream of the open reading frame of HIS3, the resulting gene fusion, if introduced into a his3 yeast strain, supported growth on histidine-lacking medium. We detected by gel retardation assay a protein specifically interacting with this fragment. All the transcriptions observed in the in vivo experiments were faithfully reproduced in a cell-free transcription system. From these results, we suggest that initiation of GAL80 transcription involves two alternative pathways; one is initiator dependent, and the other is Gal4p regulated and TATA dependent.
Sakurai
H
H
Laboratory of Molecular Genetics, Keio University School of Medicine, Tokyo, Japan.
Ohishi
T
T
Fukasawa
T
T
D001483:Base Sequence; D001665:Binding Sites; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D005809:Genes, Regulator / Q000235:genetics*; D014644:Genetic Variation; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C055002:GAL80 protein, S cerevisiae; C070633:Gal-VP16; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; D015534:Trans-Activators; D014157:Transcription Factors