Locating TATA Binding Protein in the RNA Polymerase I Pre‐Initiation Complex

ABSTRACT Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269–5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5,RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF componentRRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

Download Full-text

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.225 ◽

1996 ◽

Vol 133 (2) ◽

pp. 225-234 ◽

Cited By ~ 97

Author(s):

P Jordan ◽

M Mannervik ◽

L Tora ◽

M Carmo-Fonseca

Keyword(s):

Rna Polymerase ◽

Actinomycin D ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Rrna Genes ◽

Rrna Synthesis ◽

Rrna Transcription ◽

Polymerase I

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D-treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes.

Download Full-text

The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.557 ◽

1996 ◽

Vol 16 (2) ◽

pp. 557-563 ◽

Cited By ~ 27

Author(s):

W M Hempel ◽

A H Cavanaugh ◽

R D Hannan ◽

L Taylor ◽

L I Rothblum

Keyword(s):

Rna Polymerase ◽

Binding Protein ◽

Sodium Dodecyl ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Rrna Genes ◽

Cell Extracts ◽

Binding Factor ◽

Upstream Binding Factor ◽

Polymerase I

Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UBF from rat cell nuclear extracts and partially purified SL-1 from human cells coimmunoprecipitated with the anti-UBF antibody after mixing, indicating that the UBF-SL-1 complex can re-form. Treatment of UBF-depleted extracts with the anti-UBF antibody depleted the extracts of SL-1 activity only if UBF was added to the extract prior to the immunodepletion reaction. Furthermore, SL-1 activity could be recovered in the immunoprecipitate. Interestingly, these immunoprecipitates did not contain RNA polymerase I, as a monospecific antibody to the 194-kDa subunit of RNA polymerase I failed to detect that subunit in the immunoprecipitates. Treatment of N1S1 cell extracts with the anti-UBF antibody depleted the extracts of SL-1 activity but not TFIIIB activity, suggesting that the binding of UBF to SL-1 is specific and not solely mediated by an interaction between UBF and TATA-binding protein, which is also a component of TFIIIB. These data provide evidence that UBF and SL-1 interact.

Download Full-text

Variants of the TATA-binding protein can distinguish subsets of RNA polymerase I, II, and III promoters

Cell ◽

10.1016/0092-8674(92)90233-3 ◽

1992 ◽

Vol 69 (4) ◽

pp. 697-702 ◽

Cited By ~ 98

Author(s):

Michael C. Schultz ◽

Ronald H. Reeder ◽

Steven Hahn

Keyword(s):

Rna Polymerase ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Polymerase I

Download Full-text

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

Download Full-text

The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.235 ◽

1996 ◽

Vol 133 (2) ◽

pp. 235-246 ◽

Cited By ~ 166

Author(s):

P Roussel ◽

C André ◽

L Comai ◽

D Hernandez-Verdun

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Binding Protein ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Rdna Transcription ◽

Transcription Activity ◽

Transcription Machinery ◽

Polymerase I ◽

Active Nors

In cycling cells, the rDNAs are expressed from telophase to the end of G2 phase. The early resumption of rDNA transcription at telophase raises the question of the fate of the rDNA transcription machinery during mitosis. At the beginning of mitosis, rDNA transcription is arrested, and the rDNAs are clustered in specific chromosomal sites, the nucleolar organizer regions (NOR). In human cells, we demonstrate that the rDNA transcription machinery, as defined in vitro, is colocalized in some NORs and absent from others whatever the mitotic phase: RNA polymerase I and the RNA polymerase I transcription factors, upstream binding factor and promoter selectivity factor (as verified for TATA-binding protein and TATA-binding protein-associated factor for RNA polymerase I [110]), were colocalized in the same NORs. The RNA polymerase I complex was localized using two different antibodies recognizing the two largest subunits or only the third largest subunit, respectively. These two antibodies immunoprecipitated the RNA polymerase I complex in interphase cells as well as in mitotic cells. These results clearly indicated that the RNA polymerase I complex remained assembled during mitosis. In addition, RNA polymerase I and the transcription factors varied in the same proportions in the positive NORs, suggesting stoichiometric association of these components. The fact that the rDNA transcription machinery is not equally distributed among NORs most likely reflects the implication of the different NORs during the subsequent interphase. Indeed, we demonstrate that only positive NORs exhibit transcription activity at telophase and that the level of transcription activity is related to the amount of rDNA transcription machinery present in the NOR. We propose that assembly of rDNA transcription machinery preceding mitosis determines expression of the rDNAs at the beginning of the next cell cycle. Consequently, the association of rDNAs with the rDNA transcription machinery defines the "active" NORs and the level of activity at the transition telophase/interphase.

Download Full-text

TATA Binding Protein Can Stimulate Core-Directed Transcription by Yeast RNA Polymerase I

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.5269-5275.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 5269-5275 ◽

Cited By ~ 15

Author(s):

Pavel Aprikian ◽

Beth Moorefield ◽

Ronald H. Reeder

Keyword(s):

Rna Polymerase ◽

Binding Protein ◽

Core Promoter ◽

Contact Point ◽

Tata Binding Protein ◽

Rna Polymerase I ◽

Dual Function ◽

Polymerase I

ABSTRACT The TATA binding protein (TBP) interacts with two transcription factor complexes, upstream activating factor (UAF) and core factor (CF), to direct transcription by RNA polymerase I (polI) in the yeastSaccharomyces cerevisiae. Previous work indicates that one function of TBP is to serve as a bridge, ennabling UAF to recruit and stabilize the binding of CF (23, 24). In this work we show that, in addition to aiding recruitment, TBP also directly aids CF function. Overexpression of TBP in strains with UAF components deleted will stimulate CF-directed transcription nearly to wild-type levels in vivo. In vitro, increasing the concentration of TBP stimulates CF-directed transcription in the absence of either UAF or its DNA binding site. This dual function of TBP, serving as a critical member of a core promoter complex as well as a contact point for upstream activators, appears similar to the dual roles that TBP also plays in transcription by RNA polII.

Download Full-text