scholarly journals Evidence for the Involvement of the Glc7-Reg1 Phosphatase and the Snf1-Snf4 Kinase in the Regulation of INO1 Transcription in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 73-87 ◽  
Author(s):  
Margaret K Shirra ◽  
Karen M Arndt
Keyword(s):  
Rna Polymerase Ii ◽  
Glucose Repression ◽  
Snf1 Kinase ◽  
Glucose Levels ◽  
Recessive Mutations ◽  
Mutant Strains ◽  
Rna Polymerase Ii Transcription ◽  
Two Hybrid

AbstractBinding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.

scholarly journals Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

1989 ◽  
Vol 9 (11) ◽  
pp. 4746-4749 ◽  
Author(s):  
D I Chasman ◽  
J Leatherwood ◽  
M Carey ◽  
M Ptashne ◽  
R D Kornberg
Keyword(s):  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
In Vitro System ◽  
Transcription System ◽  
Natural Mechanism ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription ◽  
Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

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.

scholarly journals TFIIA Plays a Role in the Response to Oxidative Stress

2006 ◽  
Vol 5 (7) ◽  
pp. 1081-1090 ◽  
Author(s):  
Susan M. Kraemer ◽  
David A. Goldstrohm ◽  
Ann Berger ◽  
Susan Hankey ◽  
Sherry A. Rovinsky ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Expression Profiles ◽  
Intracellular Localization ◽  
Regulation Of Gene Expression ◽  
Wild Type ◽  
Mutant Strains

ABSTRACT To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.

scholarly journals Std1p (Msn3p) Positively Regulates the Snf1 Kinase in Saccharomyces cerevisiae

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 507-514 ◽  
Author(s):  
Sergei Kuchin ◽  
Valmik K Vyas ◽  
Ellen Kanter ◽  
Seung-Pyo Hong ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Snf1 Kinase ◽  
Glucose Signaling ◽  
Catalytic Domains ◽  
Upstream Kinase ◽  
Two Hybrid

Abstract The Snf1 protein kinase of the glucose signaling pathway in Saccharomyces cerevisiae is regulated by an autoinhibitory interaction between the regulatory and catalytic domains of Snf1p. Transitions between the autoinhibited and active states are controlled by an upstream kinase and the Reg1p-Glc7p protein phosphatase 1. Previous studies suggested that Snf1 kinase activity is also modulated by Std1p (Msn3p), which interacts physically with Snf1p and also interacts with glucose sensors. Here we address the relationship between Std1p and the Snf1 kinase. Two-hybrid assays showed that Std1p interacts with the catalytic domain of Snf1p, and analysis of mutant kinases suggested that this interaction is incompatible with the autoinhibitory interaction of the regulatory and catalytic domains. Overexpression of Std1p increased the two-hybrid interaction of Snf1p with its activating subunit Snf4p, which is diagnostic of an open, uninhibited conformation of the kinase complex. Overexpression of Std1p elevated Snf1 kinase activity in both in vitro and in vivo assays. These findings suggest that Std1p stimulates the Snf1 kinase by an interaction with the catalytic domain that antagonizes autoinhibition and promotes an active conformation of the kinase.

scholarly journals p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

1997 ◽  
Vol 17 (12) ◽  
pp. 7220-7229 ◽  
Author(s):  
L J Ko ◽  
S Y Shieh ◽  
X Chen ◽  
L Jayaraman ◽  
K Tamai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Excision Repair ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Basal Transcriptional Machinery ◽  
Nucleotide Excision ◽  
Rna Polymerase Ii Transcription

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro

1989 ◽  
Vol 9 (11) ◽  
pp. 4746-4749
Author(s):  
D I Chasman ◽  
J Leatherwood ◽  
M Carey ◽  
M Ptashne ◽  
R D Kornberg
Keyword(s):  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
In Vitro System ◽  
Transcription System ◽  
Natural Mechanism ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription ◽  
Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

scholarly journals Yeast AMP Pathway Genes Respond to Adenine through Regulated Synthesis of a Metabolic Intermediate

2001 ◽  
Vol 21 (23) ◽  
pp. 7901-7912 ◽  
Author(s):  
Karine Rébora ◽  
Christine Desmoucelles ◽  
Françoise Borne ◽  
Benoı̂t Pinson ◽  
Bertrand Daignan-Fornier
Keyword(s):  
Feedback Inhibition ◽  
Purine Nucleotide ◽  
Wild Type ◽  
Metabolic Intermediate ◽  
Mutant Strains ◽  
Extracellular Purines ◽  
Two Hybrid ◽  
Intermediate Metabolite

ABSTRACT In Saccharomyces cerevisiae, AMP biosynthesis genes (ADE genes) are transcriptionally activated in the absence of extracellular purines by the Bas1p and Bas2p (Pho2p) transcription factors. We now show that expression of theADE genes is low in mutant strains affected in the first seven steps of the pathway, while it is constitutively derepressed in mutant strains affected in later steps. Combined with epistasy studies, these results show that 5′-phosphoribosyl-4-succinocarboxamide-5-aminoimidazole (SAICAR), an intermediate metabolite of the pathway, is needed for optimal activation of the ADE genes. Two-hybrid studies establish that SAICAR is required to promote interaction between Bas1p and Bas2p in vivo, while in vitro experiments suggest that the effect of SAICAR on Bas1p-Bas2p interaction could be indirect. Importantly, feedback inhibition by ATP of Ade4p, catalyzing the first step of the pathway, appears to regulate SAICAR synthesis in response to adenine availability. Consistently, both ADE4 dominant mutations and overexpression of wild-type ADE4 lead to deregulation of ADE gene expression. We conclude that efficient transcription of yeast AMP biosynthesis genes requires interaction between Bas1p and Bas2p which is promoted in the presence of a metabolic intermediate whose synthesis is controlled by feedback inhibition of Ade4p acting as the purine nucleotide sensor within the cell.

scholarly journals Why Should DNA Topoisomerase I Have a Scaffold Activity?

Biology ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 190
Author(s):  
Francesca Di Felice ◽  
Giorgio Camilloni
Keyword(s):  
Catalytic Activity ◽  
Rna Polymerase Ii ◽  
Topoisomerase I ◽  
Ribosomal Genes ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Macromolecular Complexes ◽  
Rna Polymerase Ii Transcription

Since the early 1990s, in vitro studies have demonstrated that DNA topoisomerase I promotes RNA polymerase II transcription, acting as a cofactor, regardless of its catalytic activity. Recent studies, carried in vivo, using yeast as a model system, also demonstrate that DNA topoisomerase I is able to recruit, without the involvement of its catalytic activity, the Sir2p deacetylase on ribosomal genes thus contributes to achieve their silencing. In this review, the DNA topoisomerase I capability, acting as a scaffold protein, as well as its involvement and role in several macromolecular complexes, will be discussed, in light of several observations reported in the literature, pointing out how its role goes far beyond its well-known ability to relax DNA.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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