Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA.
A binding site for the transcription factor Abf1p was identified as an important promoter element of the gene that encodes Rpo26, a subunit common to all three yeast nuclear RNA polymerases (RNAP). Mutations in the Abf1p binding site were identified among a pool of rpo26 mutant alleles that confer synthetic lethality in combination with a temperature-sensitive mutation (rpo21-4) in the gene that encodes the largest subunit of RNAPII (Rpo21p). In the presence of the wild-type allele of RPO21 these rpo26 promoter mutations confer a cold-sensitive growth defect. Electrophoretic mobility-shift assays using purified Abf1p demonstrated that Abf1p binds to the RPO26 promoter and that the promoter mutations abolish this binding in vitro. Quantitation of the amount of RPO26 mRNA showed that mutations in the Abf1p binding site reduce the expression of RPO26 by approximately 60%. Mutations that affect Abf1p binding also result in a shift of the RPO26 transcriptional start sites to positions further upstream than normal. These results suggest that binding of the Abf1p transcription factor to the RPO26 promoter is important not only in establishing the level of transcription for this gene, but also in positioning the initiation sites of transcription.
Nouraini
S
S
Department of Genetics, Hospital for Sick Children, Toronto, Canada.
Hu
J
J
McBroom
L D
LD
Friesen
J D
JD
D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
C061886:ABF1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012321:DNA-Directed RNA Polymerases