Molecular and genetic analysis of the toxic effect of RAP1 overexpression in yeast

• Published in: Genetics (Austin) Vol. 141; no. 4; pp. 1253 - 1262
• Main Authors: Freeman, K. (SmithKline Beecham, King of Prussia, PA.), Gwadz, M, Shore, D
• Format: Journal Article
• Language: English
• Published: United States Genetics Soc America 01.12.1995; Genetics Society of America
• Subjects: ADN; Alleles; Base Sequence; Basic-Leucine Zipper Transcription Factors; Biochemistry; DNA, Fungal; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EXPRESION GENICA; EXPRESSION DES GENES; Fungal Proteins - genetics; GENE; GENES; Genes, Lethal; Genetics; GTP-Binding Proteins - genetics; INTERACCION DE GENES; INTERACTION GENIQUE; Investigations; Molecular Sequence Data; MUTACION; MUTATION; Point Mutation; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; rap GTP-Binding Proteins; Repressor Proteins - genetics; Repressor Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; TOXICIDAD; TOXICITE; Yeast
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/141.4.1253

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