scholarly journals Structure and Function of the Saccharomyces cerevisiae Sir3 BAH Domain

2006 ◽  
Vol 26 (8) ◽  
pp. 3256-3265 ◽  
Author(s):  
Jessica J. Connelly ◽  
Peihua Yuan ◽  
Hao-Chi Hsu ◽  
Zhizhong Li ◽  
Rui-Ming Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein A ◽  
Transcriptional Silencing ◽  
Structure And Function ◽  
Full Length ◽  
Left Handed ◽  
N Terminus ◽  
Bah Domain ◽  
And Function

ABSTRACT Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.

scholarly journals Mutational Analysis of the Sir3 BAH Domain Reveals Multiple Points of Interaction with Nucleosomes

2009 ◽  
Vol 29 (10) ◽  
pp. 2532-2545 ◽  
Author(s):  
Vinaya Sampath ◽  
Peihua Yuan ◽  
Isabel X. Wang ◽  
Evelyn Prugar ◽  
Fred van Leeuwen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Histone H3 ◽  
Transcriptional Silencing ◽  
In Vitro Experiments ◽  
Multiple Points ◽  
Alanine Residue ◽  
Bah Domain ◽  
Multiple Domains

ABSTRACT Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.

scholarly journals Structure-function analysis of Arabidopsis TOPLESS reveals fundamental conservation of repression mechanisms across eukaryotes

2020 ◽  
Author(s):  
Alexander R. Leydon ◽  
Wei Wang ◽  
Hardik P. Gala ◽  
Sabrina Gilmour ◽  
Samuel Juarez-Solis ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Function Analysis ◽  
Structure And Function ◽  
Environmental Cues ◽  
Auxin Response ◽  
Middle Domain ◽  
N Terminus ◽  
And Function ◽  
The Relationship ◽  
Repression Domain

SummaryThe plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle domain subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

scholarly journals Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander R Leydon ◽  
Wei Wang ◽  
Hardik P Gala ◽  
Sabrina Gilmour ◽  
Samuel Juarez-Solis ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structure And Function ◽  
Mediator Complex ◽  
Environmental Cues ◽  
Auxin Response ◽  
The Core ◽  
N Terminus ◽  
And Function ◽  
The Relationship ◽  
Repression Domain

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

scholarly journals Requirements for Transitional Endoplasmic Reticulum Site Structure and Function in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (9) ◽  
pp. 1530-1545 ◽  
Author(s):  
Polina Shindiapina ◽  
Charles Barlowe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Coat Protein ◽  
Protein Complex ◽  
Structure And Function ◽  
Site Structure ◽  
Complex Ii ◽  
And Function ◽  
Coat Protein Complex

Secretory proteins are exported from the endoplasmic reticulum (ER) at specialized regions known as the transitional ER (tER). Coat protein complex II (COPII) proteins are enriched at tER sites, although the mechanisms underlying tER site assembly and maintenance are not understood. Here, we investigated the dynamic properties of tER sites in Saccharomyces cerevisiae and probed protein and lipid requirements for tER site structure and function. Thermosensitive sec12 and sec16 mutations caused a collapse of tER sites in a manner that depended on nascent secretory cargo. Continual fatty acid synthesis was required for ER export and for normal tER site structure, whereas inhibition of sterol and ceramide synthesis produced minor effects. An in vitro assay to monitor assembly of Sec23p-green fluorescent protein at tER sites was established to directly test requirements. tER sites remained active for ∼10 min in vitro and depended on Sec12p function. Bulk phospholipids were also required for tER site structure and function in vitro, whereas depletion of phophatidylinositol selectively inhibited coat protein complex II (COPII) budding but not assembly of tER site structures. These results indicate that tER sites persist through relatively stringent treatments in which COPII budding was strongly inhibited. We propose that tER site structures are stable elements that are assembled on an underlying protein and lipid scaffold.

Structure and function of the TRP3 gene of Saccharomyces cerevisiae: Analysis of 3?- and 5?-deletions in vivo and in vitro

1984 ◽  
Vol 8 (3) ◽  
pp. 173-180 ◽  
Author(s):  
Markus Aebi ◽  
Rolf Furter ◽  
Franziska Prantl ◽  
Peter Niederberger ◽  
Ralf H�tter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structure And Function ◽  
And Function

scholarly journals Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

2008 ◽  
Vol 29 (3) ◽  
pp. 687-702 ◽  
Author(s):  
Zhonggang Hou ◽  
John R. Danzer ◽  
Liza Mendoza ◽  
Melissa E. Bose ◽  
Ulrika Müller ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Origin Recognition Complex ◽  
Full Length ◽  
Type Locus ◽  
Protein Protein Interaction ◽  
N Terminus ◽  
Bah Domain ◽  
Two Hybrid

ABSTRACTThe yeast Sir1 protein's ability to bind and silence the cryptic mating-type locusHMRarequires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bindHMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir127-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir127-149in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silenceHMRaand to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.

scholarly journals The Schizosaccharomyces pombe cho1+ Gene Encodes a Phospholipid Methyltransferase

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 553-562
Author(s):  
Margaret I Kanipes ◽  
John E Hill ◽  
Susan A Henry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Analysis ◽  
Schizosaccharomyces Pombe ◽  
Gene Disruption ◽  
Structure And Function ◽  
Biosynthetic Enzyme ◽  
Gene Encoding ◽  
Complementation Groups ◽  
Eukaryotic Organisms ◽  
And Function

Abstract The isolation of mutants of Schizosaccharomyces pombe defective in the synthesis of phosphatidylcholine via the methylation of phosphatidylethanolamine is reported. These mutants are choline auxotrophs and fall into two unlinked complementation groups, cho1 and cho2. We also report the analysis of the cho1+ gene, the first structural gene encoding a phospholipid biosynthetic enzyme from S. pombe to be cloned and characterized. The cho1+ gene disruption mutant (cho1Δ) is viable if choline is supplied and resembles the cho1 mutants isolated after mutagenesis. Sequence analysis of the cho1+ gene indicates that it encodes a protein closely related to phospholipid methyltransferases from Saccharomyces cerevisiae and rat. Phospholipid methyltransferases encoded by a rat liver cDNA and the S. cerevisiae OPI3 gene are both able to complement the choline auxotrophy of the S. pombe cho1 mutants. These results suggest that both the structure and function of the phospholipid N-methyltransferases are broadly conserved among eukaryotic organisms.

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Sign in / Sign up

Export Citation Format

Share Document