scholarly journals Transcription Activator Interactions with Multiple SWI/SNF Subunits

2002 ◽  
Vol 22 (6) ◽  
pp. 1615-1625 ◽  
Author(s):  
Kristen E. Neely ◽  
Ahmed H. Hassan ◽  
Christine E. Brown ◽  
LeAnn Howe ◽  
Jerry L. Workman
Keyword(s):  
In Vitro Transcription ◽  
Cross Linking ◽  
Dependent Manner ◽  
Transcription Activator ◽  
Activation Domains ◽  
Label Transfer ◽  
Activation Potential ◽  
Transcriptional Stimulation

ABSTRACT We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains.

scholarly journals Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535 ◽  
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

scholarly journals Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

1999 ◽  
Vol 19 (1) ◽  
pp. 855-863 ◽  
Author(s):  
Keiko Ikeda ◽  
David J. Steger ◽  
Anton Eberharter ◽  
Jerry L. Workman
Keyword(s):  
Histone Acetyltransferase ◽  
Regulation Of Transcription ◽  
Dependent Manner ◽  
Saga Complex ◽  
Histone H4 ◽  
Activation Domain ◽  
Activation Domains ◽  
Nucleosomal Array ◽  
Histone H4 Acetylation

ABSTRACT Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions.

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro

1993 ◽  
Vol 13 (3) ◽  
pp. 1719-1727
Author(s):  
C S Suen ◽  
W W Chin
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Thyroid Hormone ◽  
Promoter Activity ◽  
Hormone Receptors ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Stimulation Of ◽  
Transcriptional Stimulation

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter

1990 ◽  
Vol 10 (8) ◽  
pp. 4233-4238
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Specific Protein ◽  
Cross Linking ◽  
Dependent Manner ◽  
Hsp70 Promoter ◽  
Intimate Contact ◽  
Nuclear Extracts ◽  
Multicomponent Complex ◽  
Tata Sequence ◽  
Drosophila Embryos

A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

scholarly journals A light-induced protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide.

1995 ◽  
Vol 15 (11) ◽  
pp. 6206-6212 ◽  
Author(s):  
C Reinbothe ◽  
K Apel ◽  
S Reinbothe
Keyword(s):  
Product Formation ◽  
In Vitro Transcription ◽  
Precursor Protein ◽  
Dependent Manner ◽  
Hordeum Vulgare L ◽  
Nadph:Protochlorophyllide Oxidoreductase ◽  
Catalytically Active ◽  
Protease Treatment ◽  
Energy Dependent

The NADPH:protochlorophyllide oxidoreductase precursor protein (pPorA) of barley (Hordeum vulgare L. cv. Carina), synthesized from a full-length cDNA clone by coupling in vitro transcription and translation, is a catalytically active protein. It converts protochlorophyllide to chlorophyllide in a light- and NADPH-dependent manner. At least the pigment product of catalysis remains tightly bound to the precursor protein. The chlorophyllide-pPorA complex differs markedly from the protochlorophyllide-pPorA complex with respect to sensitivity to attack by a light-induced, nucleus-encoded, and energy-dependent protease activity of barley plastids. The pPorA-chlorophyllide complex is rapidly degraded, in contrast to pPorA-protochlorophyllide complexes containing or lacking NADPH, which are both resistant to protease treatment. Unexpectedly, pPorA devoid of its substrates or products was less sensitive to proteolysis than the pPorA-chlorophyllide complex, suggesting that both substrate binding and product formation during catalysis had caused differential changes in protein conformation.

scholarly journals UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter.

1990 ◽  
Vol 10 (8) ◽  
pp. 4233-4238 ◽  
Author(s):  
D S Gilmour ◽  
T J Dietz ◽  
S C Elgin
Keyword(s):  
Specific Protein ◽  
Cross Linking ◽  
Dependent Manner ◽  
Hsp70 Promoter ◽  
Intimate Contact ◽  
Nuclear Extracts ◽  
Multicomponent Complex ◽  
Tata Sequence ◽  
Drosophila Embryos

A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

scholarly journals A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro.

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509 ◽  
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

scholarly journals Functional Analysis of the Heat Shock Regulator HrcA of Chlamydia trachomatis

2002 ◽  
Vol 184 (23) ◽  
pp. 6566-6571 ◽  
Author(s):  
Adam C. Wilson ◽  
Ming Tan
Keyword(s):  
Heat Shock ◽  
Chlamydia Trachomatis ◽  
In Vitro Transcription ◽  
Negative Regulator ◽  
Heat Shock Gene ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Topological State ◽  
Heat Shock Gene Expression

ABSTRACT HrcA is a regulator of bacterial heat shock gene expression that binds to a cis-acting DNA element called CIRCE. It has been proposed that HrcA and CIRCE function as a repressor-operator pair. We have purified recombinant HrcA from the pathogenic bacterium Chlamydia trachomatis and have shown that it is a DNA-binding protein that functions as a negative regulator of transcription. HrcA bound specifically to the CIRCE element in a concentration-dependent manner. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter, and this repression was promoter specific. HrcA-mediated repression appears to be dependent on the topological state of the promoter, as repression on a supercoiled promoter template was greater than that on a linearized template. These results provide direct support for the role of HrcA as a transcriptional repressor in bacteria. This is the first report of the in vitro reconstitution of transcriptional regulation in Chlamydia.

Fos and jun cooperate in transcriptional regulation via heterologous activation domains

1990 ◽  
Vol 10 (10) ◽  
pp. 5532-5535
Author(s):  
C Abate ◽  
D Luk ◽  
E Gagne ◽  
R G Roeder ◽  
T Curran
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Negative Influence ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Transcriptional Stimulation

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

A tridecamer DNA sequence supports human mitochondrial RNA 3'-end formation in vitro

1988 ◽  
Vol 8 (10) ◽  
pp. 4502-4509
Author(s):  
T W Christianson ◽  
D A Clayton
Keyword(s):  
Dna Sequence ◽  
Transcription Termination ◽  
In Vitro Transcription ◽  
Mitochondrial Rna ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Transcription System ◽  
Flanking Regions

Vertebrate mitochondrial genomes contain a putative transcription termination site at the boundary between the genes for 16S rRNA and leucyl-tRNA. We have described previously an in vitro transcription system from human cells with the capacity to generate RNA 3' ends with the same map positions as those synthesized in vivo. By assaying the ability of variously truncated templates to support 3'-end formation, we demonstrated that the tridecamer sequence 5'-TGGCAGAGCCCCGG-3', contained entirely within the gene for leucyl-tRNA, is necessary to direct accurate termination. When two tridecamer sequences and their immediate flanking regions were placed in tandem, termination occurred at both promoter-proximal and promoter-distal sites. Furthermore, termination was competitively inhibited, in a concentration-dependent manner, by DNA containing the tridecamer sequence. These results suggest a modest sequence requirement for transcription termination that is contingent on a factor capable of recognizing the presence of the tridecamer DNA sequence.

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