Autoregulation of 2?m circle gene expression provides a model for maintenance of stable plasmid copy levels

Som, Tapan; Armstrong, Karen; Volkert, Frederic C.; Broach, James R.

doi:10.1016/0092-8674(88)90528-4

Cited by 125 publications

(117 citation statements)

References 40 publications

Supporting

Mentioning

107

Contrasting

Unclassified

Order By: Relevance

“…These four genes are highly expressed in the WT strain, but their expression is strongly reduced in the mutant strain. Increased amounts of Flp1 protein have previously been shown to induce 2-m recombination and thus increase the 2-m copy number (35). However, PCR analysis showed that the copy number of the 2-m plasmid in med2-S208A mutant yeast strains was indistinguishable from that of congenic WT strains.…”

Section: Discussionmentioning

confidence: 79%

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-μm plasmid

Hallberg

Polozkov

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro, and its function is conserved in all eukaryotes. Mediator interacts with both transcriptional activators and RNA polymerase II, but little is known about the mechanisms by which it operates at the molecular level. Here, we show that the cyclin-dependent kinase Srb10 interacts with, and phosphorylates, the Med2 subunit of Mediator both in vivo and in vitro. A point mutation of the single phosphorylation site in Med2 results in a strongly reduced expression of the REP1, REP2, FLP1, and RAF1 genes, which are all located on the endogenous 2-m plasmid. Combined with previous studies on the effects of SRB10͞SRB11 deletions, our data suggest that posttranslational modifications of Mediator subunits are important for regulation of gene expression.transcriptional regulation ͉ Srb11 ͉ RNA polymerase II T he Mediator complex was originally identified in Saccharomyces cerevisiae as an activity required for transcriptional activation in an in vitro transcription system reconstituted from highly purified RNA polymerase II (pol II) and general transcription factors (1, 2). Mediator was later purified to homogeneity and shown to be a complex composed of 20 subunits that interacts with the C-terminal domain of the largest pol II subunit (3, 4). More recent work has described several subunit-subunit interactions and subdomains within Mediator (5-8), and lowresolution structures of Mediator alone or in complex with pol II have been determined by three-dimensional reconstruction from electron micrographs of single particles (9, 10). These structures indicate a division of Mediator into distinct domains.In parallel with the identification of yeast Mediator, a pol II holoenzyme that comprised both Mediator subunits [a subset of general transcription factors and additional proteins (i.e., Srb8-11)] was purified (11). Nine SRB genes

show abstract

Section: Discussionmentioning

confidence: 79%

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-μm plasmid

Hallberg

Polozkov

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Plasmids used as probes for RNA analysis were as follows: for Ty, B161 (29); for TPI1, pHB59 (6); and for TUB2, pYST138 (67). The probe for indirect end labeling of the SUC2 promoter was a fragment from the 5Ј SUC2 coding region described previously (30).…”

Section: Methodsmentioning

confidence: 99%

“…Electrophoresis, transfer to GeneScreen (New England Nuclear), and hybridization analysis were performed as described previously (68). The amount of RNA in each lane was standardized by hybridization to pYST138 (67), which contains TUB2. For analysis of SUC2 mRNA, cells were grown in low-glucose media for 2.75 h as described previously (30) except that the cells were grown at 20ЊC.…”

Section: Methodsmentioning

confidence: 99%

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

Hirschhorn

Bortvin

Ricupero-Hovasse

et al. 1995

Molecular and Cellular Biology

106

View full text Add to dashboard Cite

Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N-and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.In eukaryotic cells, DNA is complexed with histones and other proteins into chromatin (see reference 75 for a review). The primary component of chromatin is the nucleosome, which consists of approximately 146 bp of DNA wrapped around an octamer of histones (two histone H2A-H2B dimers and one [H3-H4] 2 tetramer; see reference 75 for a review). A growing body of evidence from both in vivo and in vitro studies has shown that the structure of chromatin influences gene expression (see references 25 and 53 for reviews). Biochemical experiments have shown that histones can repress transcription in vitro (see reference 53 for a review) and that transcriptional activators can overcome this repression (21,41,45,79,81,82). In vivo experiments in Saccharomyces cerevisiae have shown that the loss of transcriptional activators or of activator binding sites can be suppressed by mutations in histone genes (27,30,37,57). These results suggest that one function of transcriptional activators in vivo is to antagonize chromatin-mediated repression.In vivo studies of histone mutants have also suggested that histones play a variety of roles in transcriptional regulation. Small deletions and point mutations that alter the flexible N-terminal tails of different yeast histones have been shown to cause specific changes in transcription (see reference 25 for a review). Analysis of deletions and single amino acid changes in the N-terminal region of histone H4 has demonstrated that certain changes in the H4 N terminus abolish repression of the yeast silent mating-...

show abstract

“…A decline in plasmid copy number because of rare missegregation events is corrected via DNA amplification triggered by the Flp sitespecific recombination system harbored by the plasmid (4,5). Positive and negative regulatory circuits implemented through the plasmid-coded Raf1 protein and the Rep proteins ensure quick amplification response without the danger of runaway increase in copy number (6)(7)(8).…”

mentioning

confidence: 99%

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

Huang

Chang

Cui

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The Saccharomyces cerevisiae 2-μm plasmid is a multicopy selfish genome that resides in the nucleus. The genetic organization of the plasmid is optimized for stable, high-copy propagation in hostcell populations. The plasmid's partitioning system poaches host factors, including the centromere-specific histone H3-variant Cse4 and the cohesin complex, enabling replicated plasmid copies to segregate equally in a chromosome-coupled fashion. We have characterized the in vivo chromatin topology of the plasmid partitioning locus STB in its Cse4-associated and Cse4-nonassociated states. We find that the occupancy of Cse4 at STB induces positive DNA supercoiling, with a linking difference (ΔLk) contribution estimated between +1 and +2 units. One plausible explanation for this contrary topology is the presence of a specialized Cse4-containing nucleosome with a right-handed DNA writhe at a functional STB, contrasted by a standard histone H3-containing nucleosome with a left-handed DNA writhe at a nonfunctional STB. The similarities between STB and centromere in their nucleosome signature and DNA topology would be consistent with the potential origin of the unusual point centromere of budding yeast chromosomes from the partitioning locus of an ancestral plasmid.CEN evolution | nucleosome topology | reversome T he 2-μm plasmid of Saccharomyces cerevisiae resides in the nucleus at 40 to 60 copies per cell, and propagates itself with nearly the same stability as chromosomes (1, 2). The plasmid is a benign selfish DNA element that seems to provide no advantage to its host, but poses, at its normal copy number, no serious disadvantage either. In haploid cells the plasmid is organized into a cluster of three to five foci, and segregates as a cluster (3). This effective reduction in copy number necessitates an active partitioning system, comprised of two plasmid-coded proteins, Rep1 and Rep2, and a cis-acting partitioning locus STB, to ensure equal or nearly equal plasmid segregation. A decline in plasmid copy number because of rare missegregation events is corrected via DNA amplification triggered by the Flp sitespecific recombination system harbored by the plasmid (4, 5). Positive and negative regulatory circuits implemented through the plasmid-coded Raf1 protein and the Rep proteins ensure quick amplification response without the danger of runaway increase in copy number (6-8).The Rep-STB system channels several host factors involved in chromosome segregation toward the execution of plasmid segregation. These factors include the mitotic spindle, the spindleassociated motor Kip1, the RSC2 chromatin-remodeling complex, the centromere-specific histone H3-variant Cse4 (CenH3), and the yeast cohesin complex (9-15). The de novo assembly of the plasmid-partitioning complex at STB during the G1-S window of each cell cycle (9,12,14,16) is reminiscent of the assembly of the kinetochore complex at centromeres. However, kinetochore components have not been detected at STB by ChIP (13).The assembly of the plasmid-partitioning complex culmin...

show abstract

Autoregulation of 2?m circle gene expression provides a model for maintenance of stable plasmid copy levels

Cited by 125 publications

References 40 publications

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-μm plasmid

Site-specific Srb10-dependent phosphorylation of the yeast Mediator subunit Med2 regulates gene expression from the 2-μm plasmid

A New Class of Histone H2A Mutations in Saccharomyces cerevisiae Causes Specific Transcriptional Defects In Vivo

Histone H3-variant Cse4-induced positive DNA supercoiling in the yeast plasmid has implications for a plasmid origin of a chromosome centromere

Contact Info

Product

Resources

About