scholarly journals Genetic Analysis of the Ydr1-Bur6 Repressor Complex Reveals an Intricate Balance among Transcriptional Regulatory Proteins in Yeast

2000 ◽  
Vol 20 (7) ◽  
pp. 2455-2465 ◽  
Author(s):  
Sungjoon Kim ◽  
Kettly Cabane ◽  
Michael Hampsey ◽  
Danny Reinberg
Keyword(s):  
Transcriptional Repressor ◽  
Growth Defect ◽  
Recessive Mutations ◽  
Transcriptional Regulatory ◽  
Repressor Complex ◽  
Cold Sensitive ◽  
Essential Function ◽  
Extragenic Suppressors

ABSTRACT A transcriptional repressor complex encoded by two essential genes,YDR1 and BUR6, was isolated fromSaccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1. To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo. Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor. A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1 cs) mutant led to the identification of recessive mutations in theSIN4 gene, which encodes a component of the SRB-MED complex. The sin4 alleles suppressed not onlyydr1 cs mutations but alsobur6 cs mutations. In contrast, deletion of thegal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1 cs andbur6 cs mutations, indicating that suppression is not due to general defects in the SRB-MED complex. Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of aydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background. Biochemical analysis of the SRB-MED complex from the sin4suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits. These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex.

scholarly journals Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions

2021 ◽  
Author(s):  
Jianji Chen ◽  
John Horton ◽  
Cari Sagum ◽  
Jujun Zhou ◽  
Xiaodong Cheng ◽  
...  
Keyword(s):  
Histone H3 ◽  
Transcriptional Repressor ◽  
Protein Domain ◽  
Nurd Complex ◽  
Phd Finger ◽  
Effector Domains ◽  
Methionine Residue ◽  
Repressor Complex

The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including 1) removal of the initiator methionine residue of H3 to prevent steric interference, 2) a groove where arginine-2 binds, and 3) an aromatic cage that engages methylated lysine-4. We hypothesize that  PHDs  have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ~3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.

scholarly journals Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

2018 ◽  
Author(s):  
Tommy Darrière ◽  
Michael Pilsl ◽  
Marie-Kerguelen Sarthou ◽  
Adrien Chauvier ◽  
Titouan Genty ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Pol I ◽  
Polymerase I ◽  
Extragenic Suppressors

AbstractMost transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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 Mutations affecting the tRNA-splicing endonuclease activity of Saccharomyces cerevisiae.

Genetics ◽  
1988 ◽  
Vol 118 (4) ◽  
pp. 609-617
Author(s):  
M Winey ◽  
M R Culbertson
Keyword(s):  
Growth Defect ◽  
Temperature Sensitive ◽  
Gene Products ◽  
Endonuclease Activity ◽  
Temperature Dependent ◽  
Direct Role ◽  
Trna Splicing

Abstract Two unlinked mutations that alter the enzyme activity of tRNA-splicing endonuclease have been identified in yeast. The sen1-1 mutation, which maps on chromosome 12, causes temperature-sensitive growth, reduced in vitro endonuclease activity, and in vivo accumulation of unspliced pre-tRNAs. The sen2-1 mutation does not confer a detectable growth defect, but causes a temperature-dependent reduction of in vitro endonuclease activity. Pre-tRNAs do not accumulate in sen2-1 strains. The in vitro enzyme activities of sen1-1 and sen2-1 complement in extracts from a heterozygous diploid, but fail to complement in mixed extracts from separate sen1-1 and sen2-1 haploid strains. These results suggest a direct role for SEN gene products in the enzymatic removal of introns from tRNA that is distinct from the role of other products known to affect tRNA splicing.

scholarly journals Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation

2014 ◽  
Vol 211 (5) ◽  
pp. 909-927 ◽  
Author(s):  
Adlen Foudi ◽  
Daniel J. Kramer ◽  
Jinzhong Qin ◽  
Denise Ye ◽  
Anna-Sophie Behlich ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Wide Spectrum ◽  
Transcriptional Repressor ◽  
Erythropoietin Receptor ◽  
Red Cell ◽  
Genome Wide ◽  
Cytoplasmic Maturation ◽  
Adult Bone

The zinc finger transcriptional repressor Gfi-1b is essential for erythroid and megakaryocytic development in the embryo. Its roles in the maintenance of bone marrow erythropoiesis and thrombopoiesis have not been defined. We investigated Gfi-1b’s adult functions using a loxP-flanked Gfi-1b allele in combination with a novel doxycycline-inducible Cre transgene that efficiently mediates recombination in the bone marrow. We reveal strict, lineage-intrinsic requirements for continuous adult Gfi-1b expression at two distinct critical stages of erythropoiesis and megakaryopoiesis. Induced disruption of Gfi-1b was lethal within 3 wk with severely reduced hemoglobin levels and platelet counts. The erythroid lineage was arrested early in bipotential progenitors, which did not give rise to mature erythroid cells in vitro or in vivo. Yet Gfi-1b−/− progenitors had initiated the erythroid program as they expressed many lineage-restricted genes, including Klf1/Eklf and Erythropoietin receptor. In contrast, the megakaryocytic lineage developed beyond the progenitor stage in Gfi-1b’s absence and was arrested at the promegakaryocyte stage, after nuclear polyploidization, but before cytoplasmic maturation. Genome-wide analyses revealed that Gfi-1b directly regulates a wide spectrum of megakaryocytic and erythroid genes, predominantly repressing their expression. Together our study establishes Gfi-1b as a master transcriptional repressor of adult erythropoiesis and thrombopoiesis.

scholarly journals ABAS1 from soybean is a 1R-subtype MYB transcriptional repressor that enhances ABA sensitivity

2020 ◽  
Vol 71 (10) ◽  
pp. 2970-2981
Author(s):  
Yee-Shan Ku ◽  
Meng Ni ◽  
Nacira B Muñoz ◽  
Zhixia Xiao ◽  
Annie Wing-Yi Lo ◽  
...  
Keyword(s):  
Target Gene ◽  
Transgenic Arabidopsis ◽  
Expression Patterns ◽  
Transcriptional Repressor ◽  
Aba Sensitivity ◽  
Reporter Systems ◽  
First Time ◽  
Reciprocal Expression

Abstract Transcription factors (TFs) help plants respond to environmental stresses by regulating gene expression. Up till now, studies on the MYB family of TFs have mainly focused on the highly abundant R2R3-subtype. While the less well-known 1R-subtype has been generally shown to enhance abscisic acid (ABA) sensitivity by acting as transcriptional activators, the mechanisms of their functions are unclear. Here we identified an ABA sensitivity-associated gene from soybean, ABA-Sensitive 1 (GmABAS1), of the 1R-subtype of MYB. Using the GFP-GmABAS1 fusion protein, we demonstrated that GmABAS1 is localized in the nucleus, and with yeast reporter systems, we showed that it is a transcriptional repressor. We then identified the target gene of GmABAS1 to be Glyma.01G060300, an annotated ABI five-binding protein 3 and showed that GmABAS1 binds to the promoter of Glyma.01G060300 both in vitro and in vivo. Furthermore, Glyma.01G060300 and GmABAS1 exhibited reciprocal expression patterns under osmotic stress, inferring that GmABAS1 is a transcriptional repressor of Glyma.01G060300. As a further confirmation, AtAFP2, an orthologue of Glyma.01G060300, was down-regulated in GmABAS1-transgenic Arabidopsis thaliana, enhancing the plant’s sensitivity to ABA. This is the first time a 1R-subtype of MYB from soybean has been reported to enhance ABA sensitivity by acting as a transcriptional repressor.

scholarly journals Screening of Virulence-Related Transcriptional Regulators in Streptococcus suis

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 972
Author(s):  
Liang Liu ◽  
Qiang Zhang ◽  
Zhongmin Xu ◽  
Bo Chen ◽  
Anding Zhang ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Streptococcus Suis ◽  
Transcriptional Regulators ◽  
Rna Seq ◽  
Related Factors ◽  
Transcriptional Regulatory ◽  
Mutant Strains ◽  
Whole Transcriptome

Streptococcus suis (S.suis) is an important zoonotic pathogen that causes many severe diseases in pigs and humans. Virulence-related transcriptional regulators have been widely reported in pathogenic microorganisms, but only a few have been identified in S.suis. Our aim was to screen virulence-related transcriptional regulators in S.suis. A total of 89 such genes were predicted in the S.suis genome, of which 22 were up-regulated and 18 were down-regulated during S.suis infection in mice. To evaluate the roles of these differentially expressed factors in S.suis virulence, deletion mutants were constructed, and 10 mutants were successfully obtained. Among these genes, the deletion of comR, sitR, or sxvR caused significantly decreased virulence in mice, compared to that with the wild-type strain. Moreover, the survival of ΔcomR, ΔsitR, and ΔsxvR mutant strains in blood was significantly reduced both in vitro and in vivo. Furthermore, their pro-inflammatory abilities were also obviously decreased in vivo. The regulatory mechanisms of comR, sitR, and sxvR were then analyzed by whole transcriptome RNA sequencing (RNA-Seq). Results indicated that the absence of comR induced the down-regulation of 17 virulence factors or virulence-related factors, including genes involved in the synthesis of capsules, oxidative stress tolerance, immune evasion, and cell division. Furthermore, three and two virulence factors or virulence-related factors were down-regulated upon deletion of sitR and sxvR, respectively. Thus, this study reports the discovery of three virulence-associated transcriptional regulatory factors in S.suis. These factors could ultimately be targeted to control infection caused by these bacteria.

scholarly journals Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83 Kinase Pathway by a Glycogen-Independent Mechanism

2004 ◽  
Vol 24 (1) ◽  
pp. 352-361 ◽  
Author(s):  
Heather A. Wiatrowski ◽  
Bryce J. W. van Denderen ◽  
Cristin D. Berkey ◽  
Bruce E. Kemp ◽  
David Stapleton ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Metabolic Stress ◽  
Binding Domain ◽  
Glycogen Accumulation ◽  
Snf1 Kinase ◽  
Transcriptional Regulatory ◽  
Amp Activated Protein Kinase

ABSTRACT The yeast Snf1 kinase and its mammalian ortholog, AMP-activated protein kinase (AMPK), regulate responses to metabolic stress. Previous studies identified a glycogen-binding domain in the AMPK β1 subunit, and the sequence is conserved in the Snf1 kinase β subunits Gal83 and Sip2. Here we use genetic analysis to assess the role of this domain in vivo. Alteration of Gal83 at residues that are important for glycogen binding of AMPK β1 abolished glycogen binding in vitro and caused diverse phenotypes in vivo. Various Snf1/Gal83-dependent processes were upregulated, including glycogen accumulation, expression of RNAs encoding glycogen synthase, haploid invasive growth, the transcriptional activator function of Sip4, and activation of the carbon source-responsive promoter element. Moreover, the glycogen-binding domain mutations conferred transcriptional regulatory phenotypes even in the absence of glycogen, as determined by analysis of a mutant strain lacking glycogen synthase. Thus, mutation of the glycogen-binding domain of Gal83 positively affects Snf1/Gal83 kinase function by a mechanism that is independent of glycogen binding.

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