A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival

1993 ◽  
Vol 13 (11) ◽  
pp. 6984-6991
Author(s):  
M Choder ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Ii ◽  
Stress Responses ◽  
Growth Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Transcriptional Responses ◽  
Transcriptional Changes ◽  
Substantial Decline

Cells respond to stress by altering gene expression, and these adjustments facilitate stress tolerance. Although transcriptional changes are integral to most stress responses, little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Here we report that a major role of the RNA polymerase II subunit RPB4 is to permit appropriate transcriptional responses during stress. Yeast cells lacking RPB4 have essentially wild-type growth rates at moderate temperatures (18 to 22 degrees C), but their growth rates are substantially reduced at temperatures outside this range. When subjected to a heat shock, cells lacking RPB4 rapidly lose the ability to transcribe genes and experience a dramatic loss in viability. When cells lacking RPB4 are subjected to the nutrient stress that accompanies entry into stationary phase, they also exhibit a substantial decline in mRNA synthesis and in viability relative to wild-type cells. Interestingly, the portion of RNA polymerase II molecules that contain RPB4 is small in log phase but increases substantially as cells enter stationary phase. We propose that the association of RPB4 with the other RNA polymerase II subunits increases the tolerance of the enzyme to stress.

scholarly journals A portion of RNA polymerase II molecules has a component essential for stress responses and stress survival.

1993 ◽  
Vol 13 (11) ◽  
pp. 6984-6991 ◽  
Author(s):  
M Choder ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Ii ◽  
Stress Responses ◽  
Growth Rates ◽  
Yeast Cells ◽  
Wild Type ◽  
Transcriptional Responses ◽  
Transcriptional Changes ◽  
Substantial Decline

Cells respond to stress by altering gene expression, and these adjustments facilitate stress tolerance. Although transcriptional changes are integral to most stress responses, little is known about the mechanisms that permit the transcription apparatus itself to tolerate stress. Here we report that a major role of the RNA polymerase II subunit RPB4 is to permit appropriate transcriptional responses during stress. Yeast cells lacking RPB4 have essentially wild-type growth rates at moderate temperatures (18 to 22 degrees C), but their growth rates are substantially reduced at temperatures outside this range. When subjected to a heat shock, cells lacking RPB4 rapidly lose the ability to transcribe genes and experience a dramatic loss in viability. When cells lacking RPB4 are subjected to the nutrient stress that accompanies entry into stationary phase, they also exhibit a substantial decline in mRNA synthesis and in viability relative to wild-type cells. Interestingly, the portion of RNA polymerase II molecules that contain RPB4 is small in log phase but increases substantially as cells enter stationary phase. We propose that the association of RPB4 with the other RNA polymerase II subunits increases the tolerance of the enzyme to stress.

scholarly journals CDK13 cooperates with CDK12 to control global RNA polymerase II processivity

2020 ◽  
Vol 6 (18) ◽  
pp. eaaz5041 ◽  
Author(s):  
Zheng Fan ◽  
Jennifer R. Devlin ◽  
Simon J. Hogg ◽  
Maria A. Doyle ◽  
Paul F. Harrison ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cellular Growth ◽  
Transcriptional Elongation ◽  
Transcriptional Responses ◽  
Kinase Inhibition ◽  
Homology Directed Repair ◽  
Molecular Events ◽  
Genome Wide ◽  
Transcriptional Changes

The RNA polymerase II (POLII)–driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3′ polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.

scholarly journals Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme To Transcribe at Temperature Extremes In Vitro

1998 ◽  
Vol 180 (23) ◽  
pp. 6187-6192 ◽  
Author(s):  
Sonia Rosenheck ◽  
Mordechai Choder
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Ii ◽  
Stress Responses ◽  
Chain Elongation ◽  
Temperature Extremes ◽  
Pol Ii ◽  
Cell Extracts ◽  
A Subunit

ABSTRACT Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coliprior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.

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

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 17-26 ◽  
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.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

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

2014 ◽  
Vol 25 (12) ◽  
pp. 1916-1924 ◽  
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.

Co-dependent recruitment of Ino80p and Snf2p is required for yeast CUP1 activation

2014 ◽  
Vol 92 (1) ◽  
pp. 69-75 ◽  
Author(s):  
Roshini N. Wimalarathna ◽  
Po Yun Pan ◽  
Chang-Hui Shen
Keyword(s):  
Rna Polymerase ◽  
Histone Acetylation ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Copper Toxicity ◽  
Yeast Cells ◽  
Chromatin Remodelers ◽  
Insight Into ◽  
Cup1 Promoter

In yeast, Ace1p-dependent induction of CUP1 is responsible for protecting cells from copper toxicity. Although the mechanism of yeast CUP1 induction has been studied intensively, it is still uncertain which chromatin remodelers are involved in CUP1 transcriptional activation. Here, we show that yeast cells are inviable in the presence of copper when either chromatin remodeler, Ino80p or Snf2p, is not present. This inviability is due to the lack of CUP1 expression in ino80Δ and snf2Δ cells. Subsequently, we observe that both Ino80p and Snf2p are present at the promoter and they are responsible for recruiting chromatin remodeling activity to the CUP1 promoter under induced conditions. These results suggest that they directly participate in CUP1 transcriptional activation. Furthermore, the codependent recruitment of both INO80 and SWI/SNF depends on the presence of the transcriptional activator, Ace1p. We also demonstrate that both remodelers are required to recruit RNA polymerase II and targeted histone acetylation, indicating that remodelers are recruited to the CUP1 promoter before RNA polymerase II and histone acetylases. These observations provide evidence for the mechanism of CUP1 induction. As such, we propose a model that describes novel insight into the order of events in CUP1 activation.

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth

1989 ◽  
Vol 9 (6) ◽  
pp. 2341-2349
Author(s):  
C Martin ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Gene ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Temperature Dependent ◽  
Growth Properties ◽  
Cold Sensitive ◽  
Dependent Growth ◽  
Kex2 Protease

Suppressors of a temperature-sensitive RNA polymerase II mutation were isolated to identify proteins that interact with RNA polymerase II in yeast cells. Ten independently isolated extragenic mutations that suppressed the temperature-sensitive mutation rpb1-1 and produced a cold-sensitive phenotype were all found to be alleles of a single gene, SRB1. An SRB1 partial deletion mutant was further investigated and found to exhibit several pleiotropic phenotypes. These included suppression of numerous temperature-sensitive RNA polymerase II mutations, alteration of the temperature growth range of cells containing wild-type RNA polymerase, and sterility of cells of alpha mating type. The ability of SRB1 mutations to suppress the temperature-sensitive phenotype of RNA polymerase II mutants did not extend to other temperature-sensitive mutants investigated. Isolation of the SRB1 gene revealed that SRB1 is KEX2. These results indicate that the KEX2 protease, whose only known substrates are hormone precursors, can have an important influence on RNA polymerase II and the temperature-dependent growth properties of yeast cells.

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