1. RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus
- Author
-
Mingguang Li, Nazif Maqani, Robert D. Hontz, Ryan D. Fine, Jeffrey S. Smith, Mirela Matecic, and Stephen W. Buck
- Subjects
0301 basic medicine ,Saccharomyces cerevisiae Proteins ,RNA-induced transcriptional silencing ,Transcription, Genetic ,RNA-induced silencing complex ,DNA polymerase II ,RNA polymerase II ,Saccharomyces cerevisiae ,DNA, Ribosomal ,03 medical and health sciences ,0302 clinical medicine ,Sirtuin 2 ,Transcription (biology) ,RNA Polymerase I ,Genetics ,RNA polymerase I ,Gene Silencing ,Silent Information Regulator Proteins, Saccharomyces cerevisiae ,Binding Sites ,biology ,Gene regulation, Chromatin and Epigenetics ,Molecular biology ,DNA-Binding Proteins ,RNA silencing ,030104 developmental biology ,Terminator (genetics) ,biology.protein ,030217 neurology & neurosurgery - 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.
- Published
- 2016