title: Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae.
abstract: Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.
authors.affiliation: Graduate Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, New York 10021.
authors.forename: C
authors.initials: C
authors.lastname: Liu
authors.forename: X
authors.initials: X
authors.lastname: Mao
authors.forename: A J
authors.initials: AJ
authors.lastname: Lustig
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology; D005656:Fungal Proteins / Q000502:physiology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D016615:Telomere / Q000502:physiology*; D034501:Telomere-Binding Proteins; D015534:Trans-Activators / Q000502:physiology; D020668:rap GTP-Binding Proteins
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; C084122:SIR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D034501:Telomere-Binding Proteins; D015534:Trans-Activators; C074461:RIF1 protein, S cerevisiae; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins