scholarly journals Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes.

1997 ◽  
Vol 17 (12) ◽  
pp. 6784-6793 ◽  
Author(s):  
C S Bangur ◽  
T S Pardee ◽  
A S Ponticelli
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Terminal Region ◽  
Amino Terminal ◽  
Transcription Factor Iib

The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex.

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.

Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro

1993 ◽  
Vol 13 (2) ◽  
pp. 1232-1237
Author(s):  
M E Clark ◽  
P M Lieberman ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Significant Inhibition ◽  
Pol Ii ◽  
Poliovirus Infection

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

scholarly journals Transcriptional corepression in vitro: a Mot1p-associated form of TATA-binding protein is required for repression by Leu3p.

1996 ◽  
Vol 16 (4) ◽  
pp. 1641-1648 ◽  
Author(s):  
P A Wade ◽  
J A Jaehning
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Complex Form ◽  
Tata Binding Protein ◽  
Mrna Transcription ◽  
Transcription System ◽  
Yeast Genes

Signals from transcriptional activators to the general mRNA transcription apparatus are communicated by factors associated with RNA polymerase II or the TATA-binding protein (TBP). Currently, little is known about how gene-specific transcription repressors communicate with RNA polymerase II. We have analyzed the requirements for repression by the saccharomyces cerevisiae Leu3 protein (Leu3p) in a reconstituted transcription system. We have identified a complex form of TBP which is required for communication of the repressing signal. This TFIID-like complex contains a known TBP-associated protein, Mot1p, which has been implicated in the repression of a subset of yeast genes by genetic analysis. Leu3p-dependent repression can be reconstituted with purified Mot1p and recombinant TBP. In addition, a mutation in the Mot1 gene leads to partial derepression of the Leu3p-dependent LEU2 promoter. These in vivo and in vitro observations define a role for Mot1p as a transcriptional corepressor.

scholarly journals A Divergent Transcription Factor TFIIB in Trypanosomes Is Required for RNA Polymerase II-Dependent Spliced Leader RNA Transcription and Cell Viability

2006 ◽  
Vol 5 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Jennifer B. Palenchar ◽  
Wenzhe Liu ◽  
Peter M. Palenchar ◽  
Vivian Bellofatto
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Binding Protein ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Small Nuclear Rna ◽  
Spliced Leader

ABSTRACT Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB

1993 ◽  
Vol 13 (11) ◽  
pp. 6733-6741
Author(s):  
X Xu ◽  
C Prorock ◽  
H Ishikawa ◽  
E Maldonado ◽  
Y Ito ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Basal Transcription ◽  
Transcription Factor Iib ◽  
Expression Of Genes ◽  
C Terminus ◽  
Domain Mapping

Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins.

scholarly journals Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB.

1993 ◽  
Vol 13 (11) ◽  
pp. 6733-6741 ◽  
Author(s):  
X Xu ◽  
C Prorock ◽  
H Ishikawa ◽  
E Maldonado ◽  
Y Ito ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Basal Transcription ◽  
Transcription Factor Iib ◽  
Expression Of Genes ◽  
C Terminus ◽  
Domain Mapping

Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins.

scholarly journals Direct cleavage of human TATA-binding protein by poliovirus protease 3C in vivo and in vitro.

1993 ◽  
Vol 13 (2) ◽  
pp. 1232-1237 ◽  
Author(s):  
M E Clark ◽  
P M Lieberman ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Significant Inhibition ◽  
Pol Ii ◽  
Poliovirus Infection

Host cell RNA polymerase II (Pol II)-mediated transcription is inhibited by poliovirus infection. This inhibition is correlated to a specific decrease in the activity of a chromatographic fraction which contains the transcription factor TFIID. To investigate the mechanism by which poliovirus infection results in a decrease of TFIID activity, we have analyzed a component of TFIID, the TATA-binding protein (TBP). Using Western immunoblot analysis, we show that TBP is cleaved in poliovirus-infected cells at the same time postinfection as when Pol II transcription is inhibited. Further, we show that one of the cleaved forms of TBP can be reproduced in vitro by incubating TBP with cloned, purified poliovirus encoded protease 3C. Protease 3C is a poliovirus-encoded protease that specifically cleaves glutamine-glycine bonds in the viral polyprotein. The cleavage of TBP by protease 3C occurs directly. Finally, incubation of an uninfected cell-derived TBP-containing fraction (TFIID) with protease 3C results in significant inhibition of Pol II-mediated transcription in vitro. These results demonstrate that a cellular transcription factor can be directly cleaved both in vitro and in vivo by a viral protease and suggest a role of the poliovirus proteinase 3C in host cell Pol II-mediated transcription shutoff.

scholarly journals Even-skipped Represses Transcription by Binding TATA Binding Protein and Blocking the TFIID-TATA Box Interaction

1998 ◽  
Vol 18 (7) ◽  
pp. 3771-3781 ◽  
Author(s):  
Chi Li ◽  
James L. Manley
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Direct Interaction ◽  
In Vitro Transcription ◽  
Tata Binding Protein ◽  
Tata Box ◽  
Homeodomain Protein ◽  
Necessary And Sufficient

ABSTRACT The Drosophila homeodomain protein Even-skipped (Eve) is a transcriptional repressor, and previous studies have suggested that it functions by interfering with the basal transcription machinery. Here we describe experiments indicating that the mechanism of Eve repression involves a direct interaction with the TATA binding protein (TBP) that blocks binding of TBP-TFIID to the promoter. We first compared Eve activities in in vitro transcription systems reconstituted with either all the general transcription factors or only TBP, TFIIB, TFIIF30, and RNA polymerase II. In each case, equivalent and very efficient levels of repression were observed, indicating that no factors other than those in the minimal system are required for repression. We then show that Eve can function efficiently when its recognition sites are far from the promoter and that the same regions of Eve required for repression in vivo are necessary and sufficient for in vitro repression. This includes, in addition to an Ala-Pro-rich region, residues within the homeodomain. Using GAL4-Eve fusion proteins, we demonstrate that the homeodomain plays a role in repression in addition to DNA binding, which is to facilitate interaction with TBP. Single-round transcription experiments indicate that Eve must function prior to TBP binding to the promoter, suggesting a mechanism whereby Eve represses by competing with the TATA box for TBP binding. Consistent with this, excess TATA box-containing oligonucleotide is shown to specifically and efficiently disrupt the TBP-Eve interaction. Importantly, we show that Eve binds directly to TFIID and that this interaction can also be disrupted by the TATA oligonucleotide. We conclude that Eve represses transcription via a direct interaction with TBP that blocks TFIID binding to the promoter.

scholarly journals Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

1996 ◽  
Vol 16 (5) ◽  
pp. 2350-2360 ◽  
Author(s):  
E F Michelotti ◽  
G A Michelotti ◽  
A I Aronsohn ◽  
D Levens
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Transcription In Vitro ◽  
Nuclear Ribonucleoprotein ◽  
Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

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