Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

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Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5702-5709.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5702-5709

Author(s):

J Y Sze ◽

E Remboutsika ◽

G B Kohlhaw

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Transcriptional Activation ◽

In Vitro Transcription ◽

Test System ◽

Metabolic Intermediate ◽

Mutant Forms

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

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Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5702 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5702-5709 ◽

Cited By ~ 20

Author(s):

J Y Sze ◽

E Remboutsika ◽

G B Kohlhaw

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Rna Polymerase Ii ◽

Dna Sequences ◽

Transcriptional Activation ◽

In Vitro Transcription ◽

Test System ◽

Metabolic Intermediate ◽

Mutant Forms

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 functions as a transcriptional activator only when the metabolic intermediate alpha-isopropylmalate is also present. In the absence of alpha-isopropylmalate, Leu3 causes transcription to be repressed below basal levels. We show here that different portions of the Leu3 protein are responsible for activation and repression. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding domain of the Gal4 protein created a strong cross-species activator, demonstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently developed Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Science 258:1143-1145, 1992), we show that mutant forms of the Leu3 protein that lack the activation domain still function as repressors. The shortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region of Leu3. Implications of this finding for the mechanism of repression are discussed.

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In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1989 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1989-1994 ◽

Cited By ~ 15

Author(s):

R Sen ◽

D Baltimore

Keyword(s):

B Cell ◽

Dna Sequences ◽

In Vitro Transcription ◽

Cell Extract ◽

Upstream Sequence ◽

Immunoglobulin Genes ◽

Specific Expression ◽

Sequence Element ◽

Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

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Linear DNA Does Not Form Chromatin Containing Regularly Spaced Nucleosomes

Molecular and Cellular Biology ◽

10.1128/mcb.2.12.1608 ◽

1982 ◽

Vol 2 (12) ◽

pp. 1608-1618 ◽

Cited By ~ 22

Author(s):

Janet E. Mertz

Keyword(s):

Rna Polymerase Ii ◽

Unit Length ◽

Simian Virus 40 ◽

In Vitro Transcription ◽

Micrococcal Nuclease ◽

Chromosomal Dna ◽

Histone Proteins ◽

Linear Dna ◽

Transcription System

The topological state of DNA may play a role in regulating chromatin structure and gene expression in eucaryotes. To test this hypothesis, the arrangements of nucleosomes on circular and unit-length linear simian virus 40 (SV40) DNAs incubated in nuclei ofXenopusoocytes were determined by (i) analyzing changes in the electrophoretic properties of the DNAs and (ii) examining the patterns of DNA fragments resulting from digestions with micrococcal nuclease. Whereas circular DNA became associated with nucleosomes that were arranged along the DNA at regular intervals of approximately 195 base pairs, linear DNA failed to reconstitute into chromatin containing regularly spaced nucleosomes. DNA that failed to form proper chromatin was gradually degraded, indicating that histone proteins in proper association with DNA may be the cellular component that normally protects chromosomal DNA from endonucleolytic attack. When either circular or linear DNA was incubated in an in vitro transcription system made from a whole-cell extract of HeLa cells, most of the molecules did not associate with histone proteins to form regularly spaced nucleosomes. Furthermore, linearization of mRNA-encoding DNAs, including SV40, reduces their transcriptional activity inXenopusoocytes to a level comparable to that obtained with the in vitro transcription system employed here. Therefore, proper association of DNA with appropriate cellular chromosomal factors may be a prerequisite for proper transcription by RNA polymerase II.

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Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

Molecular and Cellular Biology ◽

10.1128/mcb.23.1.335-348.2003 ◽

2003 ◽

Vol 23 (1) ◽

pp. 335-348 ◽

Cited By ~ 57

Author(s):

Mari Luz Acevedo ◽

W. Lee Kraus

Keyword(s):

Estrogen Receptor ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Steroid Receptor ◽

Estrogen Receptor Α ◽

Preinitiation Complex ◽

Transcription System ◽

Steroid Receptor Coactivator ◽

Transcription Reinitiation

ABSTRACT Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor α (ERα) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERα-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERα. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERα-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERα-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERα interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERα-dependent transcription with chromatin templates.

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Analysis of bvgR Expression in Bordetella pertussis

Journal of Bacteriology ◽

10.1128/jb.185.23.6902-6912.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6902-6912 ◽

Cited By ~ 27

Author(s):

Tod J. Merkel ◽

Philip E. Boucher ◽

Scott Stibitz ◽

Vanessa K. Grippe

Keyword(s):

Bordetella Pertussis ◽

Transcriptional Activation ◽

Northern Blot Analysis ◽

Whooping Cough ◽

Transmembrane Protein ◽

In Vitro Transcription ◽

Environmental Signals ◽

Transcription System

ABSTRACT Bordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors are dependent upon the bvg locus, which encodes three proteins: BvgA, a 23-kDa cytoplasmic protein; BvgS, a 135-kDa transmembrane protein; and BvgR, a 32-kDa protein. It is hypothesized that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator, which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by the products of the bvg locus. The repression of these genes is dependent upon the third gene, bvgR. Expression of bvgR is dependent upon the function of BvgA and BvgS. This led to the hypothesis that the binding of phosphorylated BvgA to the bvgR promoter activates the expression of bvgR. We undertook an analysis of the transcriptional activation of bvgR expression. We identified the bvgR transcript by Northern blot analysis and identified the start site of transcription by primer extension. We determined that transcriptional activation of the bvgR promoter in an in vitro transcription system requires the addition of phosphorylated BvgA. Additionally, we have identified cis-acting regions that are required for BvgA activation of the bvgR promoter by in vitro footprinting and in vivo deletion and linker scanning analyses. A model of BvgA binding to the bvgR promoter is presented.

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p300 and PCAF Act Cooperatively To Mediate Transcriptional Activation from Chromatin Templates by Notch Intracellular Domains In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.22.22.7812-7819.2002 ◽

2002 ◽

Vol 22 (22) ◽

pp. 7812-7819 ◽

Cited By ~ 174

Author(s):

Annika E. Wallberg ◽

Kia Pedersen ◽

Urban Lendahl ◽

Robert G. Roeder

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Critical Role ◽

Gene Activation ◽

In Vitro Transcription ◽

Transcription System ◽

Naked Dna ◽

Notch Receptors ◽

Intracellular Domains

ABSTRACT Ligand activation of Notch receptors leads to release of the intracellular receptor domain (Notch IC), which translocates to the nucleus and interacts with the DNA-binding protein RBP-Jκ to control expression of specific target genes. A number of proteins have been shown to interact with Notch ICs and to modulate target gene activation, but the precise function of and interplay between these factors is not known. This report investigates the Notch IC-interacting proteins, p300, PCAF, and Mastermind-like 1 (MAML1), in an in vitro transcription system with purified factors and naked DNA or chromatin templates. MAML1, RBP-Jκ, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin requires, in addition, p300, which interacts with MAML1. The transcriptional activity of p300 requires acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by MAML1 and PCAF, provide the first evidence for cooperativity between PCAF and p300 in Notch IC function, and also indicate direct effects of RBP-Jκ, Notch IC, and MAML1 on the general transcription machinery.

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