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

Genetics ◽  
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.

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 Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

2021 ◽  
Author(s):  
Ülkü Uzun ◽  
Thomas Brown ◽  
Harry Fischl ◽  
Andrew Angel ◽  
Jane Mellor
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Nucleosome Positioning ◽  
Gene Body ◽  
Precise Position ◽  
Transcription Elongation Factor

AbstractSpt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

scholarly journals The Ras/PKA Signaling Pathway of Saccharomyces cerevisiae Exhibits a Functional Interaction With the Sin4p Complex of the RNA Polymerase II Holoenzyme

Genetics ◽  
2001 ◽  
Vol 159 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Susie C Howard ◽  
Ya-Wen Chang ◽  
Yelena V Budovskaya ◽  
Paul K Herman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Ii ◽  
Signaling Pathway ◽  
Resting State ◽  
Normal Growth ◽  
Nutrient Deprivation ◽  
Ras Signaling ◽  
Rna Polymerase Ii Holoenzyme

Abstract Saccharomyces cerevisiae cells enter into the G0-like resting state, stationary phase, in response to specific types of nutrient limitation. We have initiated a genetic analysis of this resting state and have identified a collection of rye mutants that exhibit a defective transcriptional response to nutrient deprivation. These transcriptional defects appear to disrupt the control of normal growth because the rye mutants are unable to enter into a normal stationary phase upon nutrient deprivation. In this study, we examined the mutants in the rye1 complementation group and found that rye1 mutants were also defective for stationary phase entry. Interestingly, the RYE1 gene was found to be identical to SIN4, a gene that encodes a component of the yeast Mediator complex within the RNA polymerase II holoenzyme. Moreover, mutations that affected proteins within the Sin4p module of the Mediator exhibited specific genetic interactions with the Ras protein signaling pathway. For example, mutations that elevated the levels of Ras signaling, like RAS2val19, were synthetic lethal with sin4. In all, our data suggest that specific proteins within the RNA polymerase II holoenzyme might be targets of signal transduction pathways that are responsible for coordinating gene expression with cell growth.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

1990 ◽  
Vol 10 (10) ◽  
pp. 5433-5441
Author(s):  
B Y Ahn ◽  
P D Gershon ◽  
E V Jones ◽  
B Moss
Keyword(s):  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Viral Rna ◽  
In Vitro Translation ◽  
Virus Protein ◽  
Transcriptional Elongation

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

Genetic interaction between transcription elongation factor TFIIS and RNA polymerase II

1992 ◽  
Vol 12 (9) ◽  
pp. 4142-4152
Author(s):  
J Archambault ◽  
F Lacroute ◽  
A Ruet ◽  
J D Friesen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Structural Integrity ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Chemical Mutagenesis ◽  
Yeast Genome ◽  
Transcript Elongation

Little is known about the regions of RNA polymerase II (RNAPII) that are involved in the process of transcript elongation and interaction with elongation factors. One elongation factor, TFIIS, stimulates transcript elongation by binding to RNAPII and facilitating its passage through intrinsic pausing sites in vitro. In Saccharomyces cerevisiae, TFIIS is encoded by the PPR2 gene. Deletion of PPR2 from the yeast genome is not lethal but renders cells sensitive to the uracil analog 6-azauracil (6AU). Here, we show that mutations conferring 6AU sensitivity can also be isolated in the gene encoding the largest subunit of S. cerevisiae RNAPII (RPO21). A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis. All seven mutational alterations are clustered within one region of the largest subunit that is conserved among eukaryotic RNAPII. The finding that six of the seven rpo21 mutants failed to grow at elevated temperature underscores the importance of this region for the functional and/or structural integrity of RNAPII. We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS. These results are consistent with the proposal that in the rpo21 mutants, the formation of the RNAPII-TFIIS complex is rate limiting for the passage of the mutant enzyme through pausing sites. In addition to implicating a region of the largest subunit of RNAPII in the process of transcript elongation, our observations provide in vivo evidence that TFIIS is involved in transcription by RNAPII.

scholarly journals The telomeric Cdc13–Stn1–Ten1 complex regulates RNA polymerase II transcription

2019 ◽  
Vol 47 (12) ◽  
pp. 6250-6268 ◽  
Author(s):  
Olga Calvo ◽  
Nathalie Grandin ◽  
Antonio Jordán-Pla ◽  
Esperanza Miñambres ◽  
Noelia González-Polo ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Elongation Factor ◽  
Yeast Saccharomyces Cerevisiae ◽  
Replication Fork Progression ◽  
Genome Wide ◽  
Length Regulation ◽  
Transcription Regulators ◽  
Rna Polymerase Ii Transcription

Abstract Specialized telomeric proteins have an essential role in maintaining genome stability through chromosome end protection and telomere length regulation. In the yeast Saccharomyces cerevisiae, the evolutionary conserved CST complex, composed of the Cdc13, Stn1 and Ten1 proteins, largely contributes to these functions. Here, we report genetic interactions between TEN1 and several genes coding for transcription regulators. Molecular assays confirmed this novel function of Ten1 and further established that it regulates the occupancies of RNA polymerase II and the Spt5 elongation factor within transcribed genes. Since Ten1, but also Cdc13 and Stn1, were found to physically associate with Spt5, we propose that Spt5 represents the target of CST in transcription regulation. Moreover, CST physically associates with Hmo1, previously shown to mediate the architecture of S-phase transcribed genes. The fact that, genome-wide, the promoters of genes down-regulated in the ten1-31 mutant are prefentially bound by Hmo1, leads us to propose a potential role for CST in synchronizing transcription with replication fork progression following head-on collisions.

[38] RNA polymerase II transcription in vitro

1991 ◽  
pp. 545-550 ◽  
Author(s):  
Neal F. Lue ◽  
Peter M. Flanagan ◽  
Raymond J. Kelleher ◽  
Aled M. Edwards ◽  
Roger D. Kornberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription

scholarly journals RNA Polymerase II Elongation Factors of Saccharomyces cerevisiae: a Targeted Proteomics Approach

2002 ◽  
Vol 22 (20) ◽  
pp. 6979-6992 ◽  
Author(s):  
Nevan J. Krogan ◽  
Minkyu Kim ◽  
Seong Hoon Ahn ◽  
Guoqing Zhong ◽  
Michael S. Kobor ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
The Novel ◽  
Coding Region ◽  
Elongation Factors ◽  
Uncharacterized Protein ◽  
Proteomics Approach

ABSTRACT To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3′ end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

1996 ◽  
Vol 16 (5) ◽  
pp. 2350-2360 ◽  
Author(s):  
E F Michelotti ◽  
G A Michelotti ◽  
A I Aronsohn ◽  
D Levens
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Transcription In Vitro ◽  
Nuclear Ribonucleoprotein ◽  
Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

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