Within the heterochromatin of budding yeast, RNA polymerase II (RNAPII) transcription is repressed by the Sir2 deacetylase. Although heterochromatic silencing is generally thought to be due to limited accessibility of the underlying DNA, there are several reports of RNAPII and basal transcription factors within silenced regions. Analysis of the rDNA array revealed cryptic RNAPII transcription within the "nontranscribed" spacer region. These transcripts are terminated by the Nrd1/Sen1 complex and degraded by the exosome. Mutations in this pathway lead to decreased silencing and dramatic chromatin changes in the rDNA locus. Interestingly, Nrd1 mutants also show higher levels of rDNA recombination, suggesting that the cryptic RNAPII transcription might have a physiological role in regulating rDNA copy number. The Nrd1/Sen1/exosome pathway also contributes to silencing at telomeric loci. These results suggest that silencing of heterochromatic genes in Saccharomyces cerevisiae occurs at both transcriptional and posttranscriptional levels.
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http://dx.doi.org/10.1016/j.molcel.2008.01.011 | DOI Listing |
bioRxiv
January 2025
Dept. of Biochemistry, University of Colorado; Boulder, CO, 80303, USA.
CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation.
View Article and Find Full Text PDFbioRxiv
January 2025
Dept. of Biochemistry, University of Colorado, Boulder, CO, 80303, USA.
RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs and Mediator contain intrinsically-disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of RNAPII transcription at hundreds of promoters simultaneously.
View Article and Find Full Text PDFThe eukaryotic genome is broadly transcribed by RNA polymerase II (RNAPII) to produce protein-coding messenger RNAs (mRNAs) and a repertoire of non-coding RNAs (ncRNAs). Whereas RNAPII is very processive during mRNA transcription, it terminates rapidly during synthesis of many ncRNAs, particularly those that arise opportunistically from accessible chromatin at gene promoters or enhancers. The divergent fates of mRNA versus ncRNA species raise many questions about how RNAPII and associated machineries discriminate functional from spurious transcription.
View Article and Find Full Text PDFNat Commun
December 2024
Division of Experimental Parasitology, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany.
The eukaryotic nucleus exhibits a highly organized 3D genome architecture, with RNA transcription and processing confined to specific nuclear structures. While intra-chromosomal interactions, such as promoter-enhancer dynamics, are well-studied, the role of inter-chromosomal interactions remains poorly understood. Investigating these interactions in mammalian cells is challenging due to large genome sizes and the need for deep sequencing.
View Article and Find Full Text PDFbioRxiv
December 2024
Department of Biochemistry, Brandeis University, Waltham, MA 02453.
Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter.
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