Intracellular association of the protein product of the c-myc oncogene with the TATA-binding protein

The c-myc proto-oncogene encodes nuclear phosphoproteins that bind DNA in a sequence-specific fashion and appear to function as transcriptional activators. Here we demonstrate that a 40-kDa nuclear protein coimmunoprecipitated with c-Myc specifically when nuclear proteins, extracted from nuclei of exponentially growing murine B-lymphoma WEHI 231 cells by using procedures for preparation of trans-acting factors, were reacted with anti-c-Myc antibodies made against different regions of the c-Myc protein. In contrast, preparation of nuclear lysates under denaturing conditions significantly reduced this coprecipitation. Upon incubation of WEHI 231 cells with the reversible chemical cross-linking agent dithiobis(succinimidyl propionate), the 40-kDa protein could be cross-linked to c-Myc protein intracellularly. Identification of the 40-kDa protein as the TATA-binding protein (TBP) of the TFIID transcription initiation complex was made by comigration and V-8 protease mapping, which yielded identical peptide fragments upon digestion of the 40-kDa protein and material immunoprecipitated with an anti-TBP specific antibody. Furthermore, in vitro-translated TBP bound to the amino-terminal portion of c-Myc. Column chromatography of cross-linked nuclear proteins showed TBP to be in a large-molecular-weight complex with c-Myc, consistent with a transcription initiation complex. These results indicate that intracellularly, c-Myc interacts with TBP, suggesting a mechanism of interaction of this oncoprotein with the basal transcription machinery.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841-3849.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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A Human TATA Binding Protein-Related Protein with Altered DNA Binding Specificity Inhibits Transcription from Multiple Promoters and Activators

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7610 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7610-7620 ◽

Cited By ~ 66

Author(s):

Paul A. Moore ◽

Josef Ozer ◽

Moreh Salunek ◽

Gwenael Jan ◽

Dennis Zerby ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Repression ◽

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Related Protein ◽

Multiple Promoters ◽

Binding Surface

ABSTRACT The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathioneS-transferase–TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849 ◽

Cited By ~ 57

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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

Agriculture ◽

10.3390/agriculture12010081 ◽

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.

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The TATA-Binding Protein (TBP) from the Human Fungal PathogenCandida albicans Can Complement Defects in Human and Yeast TBPs

Journal of Bacteriology ◽

10.1128/jb.180.7.1771-1776.1998 ◽

1998 ◽

Vol 180 (7) ◽

pp. 1771-1776 ◽

Cited By ~ 2

Author(s):

Ping Leng ◽

Philip E. Carter ◽

Alistair J. P. Brown

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Binding Domain ◽

Amino Acid Sequences ◽

Initiation Factor ◽

Dna Binding Domain

ABSTRACT Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed theC. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15 mutation inSaccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

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Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.227 ◽

1995 ◽

Vol 15 (1) ◽

pp. 227-234 ◽

Cited By ~ 97

Author(s):

N Horikoshi ◽

A Usheva ◽

J Chen ◽

A J Levine ◽

R Weinmann ◽

...

Keyword(s):

Amino Acids ◽

Transcriptional Activation ◽

Transcriptional Repression ◽

Binding Protein ◽

Direct Interaction ◽

Tata Binding Protein ◽

Amino Terminal ◽

Terminal Domain ◽

Carboxy Terminal

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

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The Cellular TATA Binding Protein Is Required for Rep-Dependent Replication of a Minimal Adeno-Associated Virus Type 2 p5 Element

Journal of Virology ◽

10.1128/jvi.79.17.11082-11094.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11082-11094 ◽

Cited By ~ 18

Author(s):

Achille François ◽

Mickaël Guilbaud ◽

Rafi Awedikian ◽

Gilliane Chadeuf ◽

Philippe Moullier ◽

...

Keyword(s):

Virus Type ◽

Transcription Initiation ◽

Promoter Region ◽

Binding Protein ◽

Tata Binding Protein ◽

Tata Box ◽

Adeno Associated Virus

ABSTRACT The p5 promoter region of adeno-associated virus type 2 (AAV-2) is a multifunctional element involved in rep gene expression, Rep-dependent replication, and site-specific integration. We initially characterized a 350-bp p5 region by its ability to behave like a cis-acting replication element in the presence of Rep proteins and adenoviral factors. The objective of this study was to define the minimal elements within the p5 region required for Rep-dependent replication. Assays performed in transfected cells (in vivo) indicated that the minimal p5 element was composed by a 55-bp sequence (nucleotides 250 to 304 of wild-type AAV-2) containing the TATA box, the Rep binding site, the terminal resolution site present at the transcription initiation site (trs+1), and a downstream 17-bp region that could potentially form a hairpin structure localizing the trs+1 at the top of the loop. Interestingly, the TATA box was absolutely required for in vivo but dispensable for in vitro, i.e., cell-free, replication. We also demonstrated that Rep binding and nicking at the trs+1 was enhanced in the presence of the cellular TATA binding protein, and that overexpression of this cellular factor increased in vivo replication of the minimal p5 element. Together, these studies identified the minimal replication origin present within the AAV-2 p5 promoter region and demonstrated for the first time the involvement of the TATA box, in cis, and of the TATA binding protein, in trans, for Rep-dependent replication of this viral element.

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Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA

eLife ◽

10.7554/elife.22520 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 63

Author(s):

Elizabeth A Hubin ◽

Allison Fay ◽

Catherine Xu ◽

James M Bean ◽

Ruth M Saecker ◽

...

Keyword(s):

Transcription Initiation ◽

In Vivo Studies ◽

Bubble Collapse ◽

Cell Physiology ◽

Initiation Complex ◽

Transcription Bubble ◽

Transcription Initiation Complex ◽

And Function

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD.

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