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

2000 ◽  
Vol 20 (14) ◽  
pp. 5269-5275 ◽  
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.

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

1996 ◽  
Vol 133 (2) ◽  
pp. 225-234 ◽  
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.

scholarly journals In vitro definition of the yeast RNA polymerase I enhancer

1993 ◽  
Vol 13 (5) ◽  
pp. 2644-2654
Author(s):  
M C Schultz ◽  
S Y Choe ◽  
R H Reeder
Keyword(s):  
Rna Polymerase ◽  
Core Promoter ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Stable Complex ◽  
Promoter Complex ◽  
Definition Of ◽  
Polymerase I

In vitro conditions are reported under which an EcoRI-HpaI fragment of the Saccharomyces cerevisiae ribosomal gene spacer will enhance transcription from an adjacent RNA polymerase I promoter. Enhancement is largely independent of orientation and distance and is proportional to copy number. Mapping experiments reveal that two separate regions of the EcoRI-HpaI fragment are independently capable of promoter stimulation. These regions appear to correspond to elements which have been shown by previous workers to cause enhancement in vivo. Using the detergent Sarkosyl to limit the number of rounds of transcription from each promoter, we found that the degree of enhancement is similar whether one or many rounds of transcription occur. This finding supports a model in which the enhancer increases the number of stable promoter complexes but does not alter the loading of polymerase on an active promoter. Once the stable promoter complex is formed, the enhancer can be physically severed from the promoter with no loss of enhancement. Likewise, the upstream activation region of the promoter can be severed from the core promoter domain once the stable complex has been formed. These results are interpreted to mean that the enhancer functions only to assist stable complex formation and, once that is accomplished, the enhancer is dispensable.

scholarly journals In vitro definition of the yeast RNA polymerase I enhancer.

1993 ◽  
Vol 13 (5) ◽  
pp. 2644-2654 ◽  
Author(s):  
M C Schultz ◽  
S Y Choe ◽  
R H Reeder
Keyword(s):  
Rna Polymerase ◽  
Core Promoter ◽  
Ribosomal Gene ◽  
Rna Polymerase I ◽  
Stable Complex ◽  
Promoter Complex ◽  
Definition Of ◽  
Polymerase I

In vitro conditions are reported under which an EcoRI-HpaI fragment of the Saccharomyces cerevisiae ribosomal gene spacer will enhance transcription from an adjacent RNA polymerase I promoter. Enhancement is largely independent of orientation and distance and is proportional to copy number. Mapping experiments reveal that two separate regions of the EcoRI-HpaI fragment are independently capable of promoter stimulation. These regions appear to correspond to elements which have been shown by previous workers to cause enhancement in vivo. Using the detergent Sarkosyl to limit the number of rounds of transcription from each promoter, we found that the degree of enhancement is similar whether one or many rounds of transcription occur. This finding supports a model in which the enhancer increases the number of stable promoter complexes but does not alter the loading of polymerase on an active promoter. Once the stable promoter complex is formed, the enhancer can be physically severed from the promoter with no loss of enhancement. Likewise, the upstream activation region of the promoter can be severed from the core promoter domain once the stable complex has been formed. These results are interpreted to mean that the enhancer functions only to assist stable complex formation and, once that is accomplished, the enhancer is dispensable.

scholarly journals Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP

2001 ◽  
Vol 21 (7) ◽  
pp. 2292-2297 ◽  
Author(s):  
Imran Siddiqi ◽  
John Keener ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Rrna Transcription ◽  
Pol I ◽  
Mutant Strains ◽  
Polymerase I

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.

scholarly journals Nucleolin Is Required for RNA Polymerase I Transcription In Vivo

2006 ◽  
Vol 27 (3) ◽  
pp. 937-948 ◽  
Author(s):  
Brenden Rickards ◽  
S. J. Flint ◽  
Michael D. Cole ◽  
Gary LeRoy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Accessory Proteins ◽  
Naked Dna ◽  
Nucleolar Chromatin ◽  
Polymerase I

ABSTRACT Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin.

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

1996 ◽  
Vol 16 (2) ◽  
pp. 557-563 ◽  
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.

scholarly journals A DNA-binding protein is required for termination of transcription by RNA polymerase I in Xenopus laevis.

1990 ◽  
Vol 10 (6) ◽  
pp. 2793-2800 ◽  
Author(s):  
B McStay ◽  
R H Reeder
Keyword(s):  
Dna Binding ◽  
Xenopus Laevis ◽  
Rna Polymerase ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Polymerase I ◽  
Terminator Sequence

We describe a partially fractionated in vitro transcription system from Xenopus laevis for the assay of transcription termination by RNA polymerase I. Termination in vitro was found to require a specific terminator sequence in the DNA and a DNA-binding protein fraction that produces a footprint over the terminator sequence.

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