Highly Transcribed RNA Polymerase II Genes Are Impediments to Replication Fork Progression in Saccharomyces cerevisiae

Abstract The RNA polymerase (pol) III transcribes mostly short, house-keeping genes, which produce stable, non-coding RNAs. The tRNAs genes, highly transcribed by pol III in vivo are known replication fork barriers. One of the transcription factors, the PAF1C (RNA polymerase II associated factor 1 complex) is reported to associate with pol I and pol II and influence their transcription. We found low level PAF1C occupancy on the yeast pol III-transcribed genes, which is not correlated with nucleosome positions, pol III occupancy and transcription. PAF1C interacts with the pol III transcription complex and causes pol III loss from the genes under replication stress. Genotoxin exposure causes pol III but not Paf1 loss from the genes. In comparison, Paf1 deletion leads to increased occupancy of pol III, γ-H2A and DNA pol2 in gene-specific manner. Paf1 restricts the accumulation of pol III by influencing the pol III pause on the genes, which reduces the pol III barrier to the replication fork progression.

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Determinants of Replication-Fork Pausing at tRNA Genes in Saccharomyces cerevisiae

Genetics ◽

10.1534/genetics.120.303092 ◽

2020 ◽

Vol 214 (4) ◽

pp. 825-838

Author(s):

Rani Yeung ◽

Duncan J. Smith

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Repair ◽

Transcription Factor ◽

Rna Polymerase ◽

Replication Fork ◽

Rna Polymerase Iii ◽

Trna Genes ◽

Link Type ◽

Replication Fork Progression ◽

Polymerase Iii

Transfer RNA (tRNA) genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae. tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

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Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.3.737 ◽

1996 ◽

Vol 142 (3) ◽

pp. 737-747 ◽

Cited By ~ 7

Author(s):

Jacques Archambault ◽

David B Jansma ◽

James D Friesen

Keyword(s):

Saccharomyces Cerevisiae ◽

Steady State ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Reporter Gene ◽

Growth Medium ◽

Temperature Sensitivity ◽

Slow Growth ◽

Yeast Saccharomyces Cerevisiae ◽

Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

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Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)50101-0 ◽

1992 ◽

Vol 267 (32) ◽

pp. 23376-23382 ◽

Cited By ~ 3

Author(s):

M.H. Sayre ◽

H Tschochner ◽

R.D. Kornberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Initiation Factors

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The rye Mutants Identify a Role for Ssn/Srb Proteins of the RNA Polymerase II Holoenzyme During Stationary Phase Entry in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.17 ◽

2001 ◽

Vol 157 (1) ◽

pp. 17-26 ◽

Cited By ~ 3

Author(s):

Ya-Wen Chang ◽

Susie C Howard ◽

Yelena V Budovskaya ◽

Jasper Rine ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Stationary Phase ◽

Yeast Cell ◽

Rna Polymerase Ii ◽

Transcriptional Response ◽

Nutrient Deprivation ◽

Ras Signaling ◽

Rna Polymerase Ii Holoenzyme

Abstract Saccharomyces cerevisiae cells enter into a distinct resting state, known as stationary phase, in response to specific types of nutrient deprivation. We have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. In wild-type cells, YGP1 levels increased during the growth arrest caused by nutrient deprivation or inactivation of the Ras signaling pathway. In contrast, the levels of YGP1 and related genes were significantly elevated in the rye mutants during log phase growth. The rye defects were not specific to this YGP1 response as these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation. These data indicated that the RYE genes might encode important regulators of yeast cell growth. Interestingly, three of the RYE genes encoded the Ssn/Srb proteins, Srb9p, Srb10p, and Srb11p, which are associated with the RNA polymerase II holoenzyme. Thus, the RNA polymerase II holoenzyme may be a target of the signaling pathways responsible for coordinating yeast cell growth with nutrient availability.

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Genetic interactions between an RNA polymerase II phosphatase and centromeric elements in Saccharomyces cerevisiae

Molecular Genetics and Genomics ◽

10.1007/s00438-004-1009-5 ◽

2004 ◽

Vol 271 (5) ◽

pp. 603-615 ◽

Cited By ~ 3

Author(s):

E. Pierstorff ◽

C. M. Kane

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genetic Interactions

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The Ras/PKA Signaling Pathway May Control RNA Polymerase II Elongation via the Spt4p/Spt5p Complex in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/165.3.1059 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1059-1070

Author(s):

Susie C Howard ◽

Arelis Hester ◽

Paul K Herman

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Growth ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Signaling Pathway ◽

Elongation Factor ◽

Ras Signaling ◽

Elongation Step ◽

Rna Polymerase Ii Transcription

Abstract The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

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Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0591 ◽

2014 ◽

Vol 25 (12) ◽

pp. 1916-1924 ◽

Cited By ~ 3

Author(s):

David Öling ◽

Rehan Masoom ◽

Kristian Kvint

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Life Span ◽

Rna Polymerase Ii ◽

Ribosomal Dna ◽

Genetic Evidence ◽

Wild Type ◽

Replicative Life Span ◽

Unequal Recombination

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes.

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Genetic exploration of interactive domains in RNA polymerase II subunits

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.1908-1914.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 1908-1914

Author(s):

C Martin ◽

S Okamura ◽

R Young

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Temperature Sensitive ◽

Suppressor Mutations ◽

Region I ◽

Temperature Sensitive Mutation ◽

Allele Specific ◽

Separate Protein

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

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