Molecular and genetic analysis of the toxic effect of RAP1 overexpression in yeast.
Rap1p is a context-dependent regulatory protein in yeast that functions as a transcriptional activator of many essential genes, including those encoding ribosomal proteins and glycolytic enzymes. Rap1p also participates in transcriptional silencing at HM mating-type loci and telomeres. Overexpression of RAP1 strongly inhibits cell growth, perhaps by interfering with essential transcriptional activation functions within the cell. Here we report a molecular and genetic analysis of the toxic effect of RAP1 overexpression. We show that toxicity does not require the previously defined Rap1p activation and silencing domains, but instead is dependent upon the DNA-binding domain and an adjacent region of unknown function. Point mutations were identified in the DNA-binding domain that relieve the toxic effect of overexpression. Two of these mutations can complement a RAP1 deletion yet cause growth defects and altered DNA-binding properties in vitro. However, a small deletion of the adjacent (downstream) region that abolishes overexpression toxicity has, by itself, no apparent effect on growth or DNA binding. SKO1/ACR1, which encodes a CREB-like repressor protein in yeast, was isolated as a high copy suppressor of the toxicity caused by RAP1 overexpression. Models related to the regulation of Rap1p activity are discussed.
Freeman
K
K
Department of Microbiology, College of Physicians and Surgeons of Columbia University, New York 10032, USA.
Gwadz
M
M
Shore
D
D
D000483:Alleles; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D004271:DNA, Fungal; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics*; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D017354:Point Mutation; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D020668:rap GTP-Binding Proteins
D050976:Basic-Leucine Zipper Transcription Factors; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; C077832:SKO1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins