The ZEBRA activation domain: modular organization and mechanism of action

1993 ◽  
Vol 13 (11) ◽  
pp. 7045-7055
Author(s):  
T Chi ◽  
M Carey
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Response ◽  
Nuclear Extract ◽  
Modular Organization ◽  
Modular Architecture ◽  
Activation Domain ◽  
Kinetic Measurements ◽  
Complex Assembly ◽  
Amino Terminal

An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored. ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites. Our data show that ZEBRA contains four modules that contribute to its potency in vitro. The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites. Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly. Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor. We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.

scholarly journals The ZEBRA activation domain: modular organization and mechanism of action.

1993 ◽  
Vol 13 (11) ◽  
pp. 7045-7055 ◽  
Author(s):  
T Chi ◽  
M Carey
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Response ◽  
Nuclear Extract ◽  
Modular Organization ◽  
Modular Architecture ◽  
Activation Domain ◽  
Kinetic Measurements ◽  
Complex Assembly ◽  
Amino Terminal

An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers. The regulatory significance of this parallel organization has not been systematically explored. We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA. We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored. ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites. Our data show that ZEBRA contains four modules that contribute to its potency in vitro. The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites. Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly. Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor. We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

2004 ◽  
Vol 24 (7) ◽  
pp. 2863-2874 ◽  
Author(s):  
Thomas C. Tubon ◽  
William P. Tansey ◽  
Winship Herr
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Terminal Region ◽  
General Role ◽  
Pol Ii ◽  
Amino Terminal ◽  
Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

scholarly journals The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

1998 ◽  
Vol 18 (3) ◽  
pp. 1711-1724 ◽  
Author(s):  
Connie M. Drysdale ◽  
Belinda M. Jackson ◽  
Richard McVeigh ◽  
Edward R. Klebanow ◽  
Yu Bai ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Adapter Proteins ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Cell Extracts ◽  
Tata Box Binding Protein ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathioneS-transferase (GST)–Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive effects of these mutations on binding of coactivator proteins correlated with their cumulative effects on transcriptional activation by Gcn4p in vivo, particularly with Ada3p, suggesting that recruitment of these coactivator complexes to the promoter is a cardinal function of the Gcn4p activation domain. As judged by immunoprecipitation analysis, components of the mediator were not associated with constituents of TFIID and Adap-Gcn5p in the extracts, implying that GST-Gcn4p interacted with the mediator independently of these other coactivators. Unexpectedly, a proportion of Ada2p coimmunoprecipitated with yTAFII90, and the yTAFII20, -60, and -90 proteins were coimmunoprecipitated with Ada3p, revealing a stable interaction between components of TFIID and the Adap-Gcn5p complex. Because GST-Gcn4p did not bind specifically to highly purified TFIID, Gcn4p may interact with TFIID via the Adap-Gcn5p complex or some other adapter proteins. The ability of Gcn4p to interact with several distinct coactivator complexes that are physically and genetically linked to TATA box-binding protein can provide an explanation for the observation that yTAFII proteins are dispensable for activation by Gcn4p in vivo.

scholarly journals Involvement of Negative Cofactor NC2 in Active Repression by Zinc Finger-Homeodomain Transcription Factor AREB6

1998 ◽  
Vol 18 (1) ◽  
pp. 10-18 ◽  
Author(s):  
Keiko Ikeda ◽  
Jörn-Peter Halle ◽  
Gertraud Stelzer ◽  
Michael Meisterernst ◽  
Kiyoshi Kawakami
Keyword(s):  
Amino Acids ◽  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Zinc Finger ◽  
Transcriptional Repression ◽  
Consensus Sequence ◽  
Nuclear Extract ◽  
Transcription System ◽  
Repression Domain

ABSTRACT The transcription factor AREB6 contains a homeodomain flanked by two clusters of Krüppel type C2H2 zinc fingers. AREB6 binds to the E-box consensus sequence, CACCTGT, through either the N- or the C-terminal zinc finger cluster. To gain insights into the molecular mechanism by which AREB6 activates and represses gene expression, we analyzed the domain structure of AREB6 in the context of a heterologous DNA-binding domain by transient-transfection assays. The C-terminal region spanning amino acids 1011 to 1124 was identified as a conventional acidic activation domain. The region containing amino acids 754 to 901, which was identified as a repression domain, consists of 40% hydrophobic amino acids displaying no sequence similarities to other known repression domains. This region repressed transcription in vitro in a HeLa nuclear extract but not in reconstituted transcription systems consisting of transcription factor IID (TFIID), TFIIB, TFIIE, TFIIH/F, and RNA polymerase II. The addition of recombinant negative cofactor NC2 (NC2α/DRAP1 and NC2β/Dr1) to the reconstituted transcription system restored the activity of the AREB6 repression domain. We further demonstrated interactions between the AREB6 repression domain and NC2α in yeast two-hybrid assay. Our findings suggest a mechanism of transcriptional repression that is mediated by the general cofactor NC2.

scholarly journals A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors.

1995 ◽  
Vol 129 (4) ◽  
pp. 899-908 ◽  
Author(s):  
K M Neugebauer ◽  
J A Stolk ◽  
M B Roth
Keyword(s):  
Rna Polymerase Ii ◽  
Active Sites ◽  
Nuclear Extract ◽  
Sr Proteins ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Structural Element ◽  
Small Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The removal of introns from eukaryotic pre-mRNA occurs in a large ribonucleoprotein complex called the spliceosome. We have generated a monoclonal antibody (mAb 16H3) against four of the family of six SR proteins, known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins, SRp20, SRp40, SRp55, and SRp75, mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription which suggests their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope was mapped to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, we propose that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors.

scholarly journals Inhibition of Androgen Receptor-Mediated Transcription by Amino-Terminal Enhancer of split

2001 ◽  
Vol 21 (14) ◽  
pp. 4614-4625 ◽  
Author(s):  
Xin Yu ◽  
Peng Li ◽  
Robert G. Roeder ◽  
Zhengxin Wang
Keyword(s):  
Androgen Receptor ◽  
Nuclear Extract ◽  
Basal Transcription ◽  
Hela Nuclear Extract ◽  
Protein Protein Interaction ◽  
Transcription System ◽  
Amino Terminal ◽  
Two Hybrid ◽  
Enhancer Of Split

ABSTRACT A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors. Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays. AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract. These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery.

scholarly journals ScRpb4, Encoding an RNA Polymerase Subunit from Sugarcane, Is Ubiquitously Expressed and Resilient to Changes in Response to Stress Conditions

Agriculture ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 81
Author(s):  
Taehoon Kim ◽  
Fábio Ometto Dias ◽  
Agustina Gentile ◽  
Marcelo Menossi ◽  
Kevin Begcy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Response ◽  
Stress Conditions ◽  
Initiation Complex ◽  
Initial Characterization ◽  
Transcription Initiation Complex

RNA polymerase II is an essential multiprotein complex that transcribes thousands of genes, being a fundamental component of the transcription initiation complex. In eukaryotes, RNA polymerase II is formed by a 10-multisubunit conserved core complex, and two additional peripheral subunits, Rpb4 and Rpb7, form the Rpb4/7 subcomplex. Although transcription is vital for cell and organismal viability, little is known about the transcription initiation complex in sugarcane. An initial characterization of the sugarcane RNA polymerase subunit IV (ScRpb4) was performed. Our results demonstrate that ScRpb4 is evolutionarily conserved across kingdoms. At the molecular level, ScRpb4 expression was found in vegetative and reproductive tissues. Furthermore, the expression of ScRpb4 remained stable under various stress conditions, most likely to ensure a proper transcriptional response. Optimal conditions to express ScRpb4 in vitro for further studies were also identified. In this study, an initial characterization of the sugarcane polymerase II subunit IV is presented. Our results open the window to more specific experiments to study ScRpb4 function, for instance, crystal structure determination and pull-down assays as well as their function under biotic and abiotic stresses.

scholarly journals INCENP Centromere and Spindle Targeting: Identification of Essential Conserved Motifs and Involvement of Heterochromatin Protein HP1

1998 ◽  
Vol 143 (7) ◽  
pp. 1763-1774 ◽  
Author(s):  
Alexandra M. Ainsztein ◽  
Stefanie E. Kandels-Lewis ◽  
Alastair M. Mackay ◽  
William C. Earnshaw
Keyword(s):  
Amino Acid ◽  
Centromeric Heterochromatin ◽  
Modular Organization ◽  
Physical Interaction ◽  
Sequence Motifs ◽  
Amino Acid Motif ◽  
Heterochromatin Protein ◽  
Amino Terminal ◽  
Vitro Binding

The inner centromere protein (INCENP) has a modular organization, with domains required for chromosomal and cytoskeletal functions concentrated near the amino and carboxyl termini, respectively. In this study we have identified an autonomous centromere- and midbody-targeting module in the amino-terminal 68 amino acids of INCENP. Within this module, we have identified two evolutionarily conserved amino acid sequence motifs: a 13–amino acid motif that is required for targeting to centromeres and transfer to the spindle, and an 11–amino acid motif that is required for transfer to the spindle by molecules that have targeted previously to the centromere. To begin to understand the mechanisms of INCENP function in mitosis, we have performed a yeast two-hybrid screen for interacting proteins. These and subsequent in vitro binding experiments identify a physical interaction between INCENP and heterochromatin protein HP1Hsα. Surprisingly, this interaction does not appear to be involved in targeting INCENP to the centromeric heterochromatin, but may instead have a role in its transfer from the chromosomes to the anaphase spindle.

scholarly journals The WW Domain-Containing Proteins Interact with the Early Spliceosome and Participate in Pre-mRNA Splicing In Vivo

2004 ◽  
Vol 24 (20) ◽  
pp. 9176-9185 ◽  
Author(s):  
Kai-Ti Lin ◽  
Ruei-Min Lu ◽  
Woan-Yuh Tarn
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Ww Domain ◽  
Ww Domains ◽  
Small Nuclear Ribonucleoprotein

ABSTRACT A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3′ part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3′ splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.

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