An Interaction between the N-terminal Region and the Core Domain of Yeast TFIIB Promotes the Formation of TATA-binding Protein-TFIIB-DNA Complexes

Abstract The TATA-binding protein (TBP) is composed of a highly conserved core domain sufficient for TATA-element binding and preinitiation complex formation as well as a highly divergent N-terminal region that is dispensable for yeast cell viability. In vitro, removal of the N-terminal region domain enhances TBP-TATA association and TBP dimerization. Here, we examine the effects of truncation of the N-terminal region in the context of yeast TBP mutants with specific defects in DNA binding and in interactions with various proteins. For a subset of mutations that disrupt DNA binding and the response to transcriptional activators, removal of the N-terminal domain rescues their transcriptional defects. By contrast, deletion of the N-terminal region is lethal in combination with mutations on a limited surface of TBP. Although this surface is important for interactions with TFIIA and Brf1, TBP interactions with these two factors do not appear to be responsible for this dependence on the N-terminal region. Our results suggest that the N-terminal region of TBP has at least two distinct functions in vivo. It inhibits the interaction of TBP with TATA elements, and it acts positively in combination with a specific region of the TBP core domain that presumably interacts with another protein(s).

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Faculty Opinions recommendation of The TATA-binding protein core domain in solution variably bends TATA sequences via a three-step binding mechanism.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1157880.619132 ◽

2009 ◽

Author(s):

Herschel Wade

Keyword(s):

Binding Protein ◽

Tata Binding Protein ◽

Binding Mechanism ◽

Core Domain ◽

Protein Core

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A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E

Biochemical Journal ◽

10.1042/bj20101481 ◽

2011 ◽

Vol 441 (1) ◽

pp. 237-245 ◽

Cited By ~ 26

Author(s):

Keum Soon Paku ◽

Yu Umenaga ◽

Tsunego Usui ◽

Ai Fukuyo ◽

Atsuo Mizuno ◽

...

Keyword(s):

Amino Acid ◽

Binding Protein ◽

Tight Binding ◽

Terminal Region ◽

Dynamics Simulation ◽

Initiation Factor ◽

Binding Region ◽

The Core ◽

C Terminus ◽

Conserved Region

Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1–3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.

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Variations in Intracellular Levels of TATA Binding Protein Can Affect Specific Genes by Different Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.00809-07 ◽

2007 ◽

Vol 28 (1) ◽

pp. 83-92 ◽

Cited By ~ 4

Author(s):

Stephanie D. Bush ◽

Patricia Richard ◽

James L. Manley

Keyword(s):

Binding Protein ◽

Core Promoter ◽

Tata Binding Protein ◽

Tata Box ◽

Wild Type ◽

The Core ◽

Mitotic Delay ◽

Reduced Activity ◽

Dt40 Cells ◽

Repressor Element

ABSTRACT We previously showed that reduced intracellular levels of the TATA binding protein (TBP), brought about by tbp heterozygosity in DT40 cells, resulted in a mitotic delay reflecting reduced expression of the mitotic regulator cdc25B but did not significantly affect overall transcription. Here we extend these findings in several ways. We first provide evidence that the decrease in cdc25B expression reflects reduced activity of the cdc25B core promoter in the heterozygous (TBP-het) cells. Strikingly, mutations in a previously described repressor element that overlaps the TATA box restored promoter activity in TBP-het cells, supporting the idea that the sensitivity of this promoter to TBP levels reflects a competition between TBP and the repressor for DNA binding. To determine whether cells might have mechanisms to compensate for fluctuations in TBP levels, we next examined expression of the two known vertebrate TBP homologues, TLP and TBP2. Significantly, mRNAs encoding both were significantly overexpressed relative to levels observed in wild-type cells. In the case of TLP, this was shown to reflect regulation of the core promoter by both TBP and TLP. Together, our results indicate that variations in TBP levels can affect the transcription of specific promoters in distinct ways, but overall transcription may be buffered by corresponding alterations in the expression of TBP homologues.

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Recruitment of CBP/p300, TATA-Binding Protein, and S8 to Distinct Regions at the N Terminus of Adenovirus E1A

Journal of Virology ◽

10.1128/jvi.79.9.5594-5605.2005 ◽

2005 ◽

Vol 79 (9) ◽

pp. 5594-5605 ◽

Cited By ~ 32

Author(s):

Mozhgan Rasti ◽

Roger J. A. Grand ◽

Joe S. Mymryk ◽

Phillip H. Gallimore ◽

Andrew S. Turnell

Keyword(s):

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Binding Protein ◽

Cellular Transformation ◽

Tata Binding Protein ◽

Transformation Process ◽

Terminal Region ◽

Site Directed Mutagenesis

ABSTRACT The N-terminal region of the adenovirus (Ad) 12S E1A gene product targets several cellular proteins that are essential for the induction of S phase, cellular immortalization, cellular transformation, transcriptional repression, and transcriptional activation. The precise binding sites for these proteins, however, remain to be resolved. We therefore undertook an extensive site-directed mutagenesis approach to generate specific point mutants and to precisely map the binding sites for CBP, p300, TATA-binding protein (TBP), S4, S8, hGcn5, P/CAF, and Ran within the first 30 amino acids of the Ad5 12S E1A protein. We determined that although common residues within the N-terminal region can form partial binding sites for these proteins, point mutants were also generated that could discriminate between binding sites. These data indicate that AdE1A can target each of these proteins individually through distinct binding sites. It was evident, however, that the mutation of specific hydrophobic residues typically had the greatest effect upon AdE1A's ability to bind individual partners. Indeed, the mutation of L at positions 19 and 20 eliminated the ability of AdE1A to interact with any of the N-terminal binding proteins studied here. Interestingly, although TBP and S8 or CBP/p300 can exist as functional complexes, RNA interference revealed that the recruitment of either TBP, S8, or CBP/p300 to AdE1A was not dependent upon the expression of the other proteins. These data further indicate that AdE1A can target individual partner proteins in vivo and that it does not necessarily recruit these proteins indirectly as components of larger macromolecular complexes. Finally, we took advantage of the fine-mapping data to ascertain which proteins were targeted during the transformation process. Consistent with previous studies, CBP/p300 was found to be targeted by AdE1A during this process, although our data suggest that binding to other N-terminal proteins is also important for transformation.

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Domains of the Brf component of RNA polymerase III transcription factor IIIB (TFIIIB): functions in assembly of TFIIIB-DNA complexes and recruitment of RNA polymerase to the promoter.

Molecular and Cellular Biology ◽

10.1128/mcb.17.9.5299 ◽

1997 ◽

Vol 17 (9) ◽

pp. 5299-5306 ◽

Cited By ~ 34

Author(s):

G A Kassavetis ◽

C Bardeleben ◽

A Kumar ◽

E Ramirez ◽

E P Geiduschek

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Binding Protein ◽

Rna Polymerase Iii ◽

Tata Binding Protein ◽

Terminal Segment ◽

Dna Complexes ◽

Polymerase Iii ◽

Transcription Factor Iiib ◽

Derivatives Of

Saccharomyces cerevisiae transcription factor IIIB (TFIIIB) is composed of three subunits: the TATA-binding protein, the TFIIB-related protein Brf, and B". TFIIIB, which is brought to RNA polymerase III-transcribed genes indirectly through interaction with DNA-bound TFIIIC or directly through DNA recognition by the TATA-binding protein, in turn recruits RNA polymerase III to the promoter. N-terminally deleted derivatives of Brf have been examined for their ability to interact with DNA-bound TFIIIC and with the other components of TFIIIB and for participation in transcription. Brf(165-596), lacking 164 N-proximal TFIIB-homologous amino acids, is competent to participate in the assembly of TFIIIB-DNA complexes and in TFIIIC-independent transcription. Even deletion of the entire TFIIB-homologous half of the protein, as in Brf(317-596) and Brf(352-596), allows some interaction with DNA-bound TBP and with the B" component of TFIIIB to be retained. The function of Brf(165-596) in transcription has also been examined in the context of B" with small internal deletions. The ability of Brf with this sizable N-terminal segment deleted to function in TFIIIC-independent transcription requires segments of B" that are individually indispensable although required on an either/or basis, in the context of complete Brf. These findings suggest a functional complementarity and reciprocity between the Brf and B" components of TFIIIB.

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Solution structure of the c-terminal core domain of human TFIIB: Similarity to cyclin A and interaction with TATA-binding protein

Cell ◽

10.1016/0092-8674(95)90483-2 ◽

1995 ◽

Vol 82 (5) ◽

pp. 857-867 ◽

Cited By ~ 88

Author(s):

Stefan Bagby ◽

Sungjoon Kim ◽

Edio Maldonado ◽

Kit I Tong ◽

Danny Reinberg ◽

...

Keyword(s):

Solution Structure ◽

Binding Protein ◽

Cyclin A ◽

Tata Binding Protein ◽

Core Domain

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The conserved core domain of the human TATA binding protein is sufficient to assemble the multisubunit RNA polymerase I-specific transcription factor SL1.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.17.8229 ◽

1994 ◽

Vol 91 (17) ◽

pp. 8229-8233 ◽

Cited By ~ 11

Author(s):

U. Rudloff ◽

D. Eberhard ◽

I. Grummt

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Core Domain ◽

Specific Transcription Factor ◽

Polymerase I ◽

Conserved Core

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The yeast TATA-binding protein (TBP) core domain assembles with human TBP-associated factors into a functional TFIID complex.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.534 ◽

1995 ◽

Vol 15 (1) ◽

pp. 534-539 ◽

Cited By ~ 9

Author(s):

Q Zhou ◽

A J Berk

Keyword(s):

Rna Polymerase Ii ◽

Associated Factors ◽

Binding Protein ◽

Tata Binding Protein ◽

Stable Complex ◽

Core Domain ◽

Cell Extracts ◽

Hela Cell Lines ◽

Tfiid Complex

In mammalian and Drosophila cells, the central RNA polymerase II general transcription factor TFIID is a multisubunit complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) bound to the conserved TBP carboxy-terminal core domain. TBP also associates with alternative TAFs in these cells to form general transcription factors required for initiation by RNA polymerases I and III. Although extracts of human HeLa cells contain little TBP that is not associated with TAFs, free TBP is readily isolated from yeast cell extracts. However, recent studies indicate that yeast TBP can also interact with other yeast polypeptides to form multiprotein complexes. We established stable human HeLa cell lines expressing yeast TBP and several yeast-human TBP hybrids to study TBP-TAF interactions. We found that the yeast TBP core domain assembles with a complete set of human TAFs into a stable TFIID complex that can support activated transcription in vitro. The fact that the yeast TBP core, which differs from human TBP core in approximately 20% of its amino acid residues, has the structural features required to form a stable complex with human TAFs implies that Saccharomyces cerevisiae probably contains TAFs that are structurally and functionally analogous to human TAFs. Surprisingly, the non-conserved amino terminus of yeast TBP inhibited association between the yeast core domain and human TAFs.

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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.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.6784 ◽

1997 ◽

Vol 17 (12) ◽

pp. 6784-6793 ◽

Cited By ~ 40

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.

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