title: RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus.
abstract: RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Buck
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Nazif
authors.initials: N
authors.lastname: Maqani
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Mirela
authors.initials: M
authors.lastname: Matecic
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Hontz
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Fine
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
authors.forename: Mingguang
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA jss5y@virginia.edu.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Smith
mesh_terms: D001665:Binding Sites; D004275:DNA, Ribosomal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D020868:Gene Silencing; D012318:RNA Polymerase I / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics; D056565:Sirtuin 2 / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C105579:FOB1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D012318:RNA Polymerase I; C087778:SIR2 protein, S cerevisiae; D056565:Sirtuin 2