Stable transcription complex on a class III gene in a minichromosome.

We have constructed recombinant simian virus 40 molecules containing Xenopus 5S RNA and tRNA genes. Recombinant minichromosomes containing these genes were isolated to study the interaction of RNA polymerase III transcription factors with these model chromatin templates. Minichromosomes containing a tRNAMet gene can be isolated in a stable complex with transcription factors (IIIB and IIIC) and are active in vitro templates for purified RNA polymerase III. In contrast, minichromosomes containing a 5S RNA gene are refractory to transcription by purified RNA polymerase III in either the absence or the presence of other factors.

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Upstream basal promoter element important for exclusive RNA polymerase III transcription of the EBER 2 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2655 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2655-2665 ◽

Cited By ~ 18

Author(s):

J G Howe ◽

M D Shu

Keyword(s):

Rna Polymerase ◽

Consensus Sequence ◽

Rna Polymerase Iii ◽

Class Ii ◽

Transcription Unit ◽

Tata Box ◽

Promoter Element ◽

Class Iii ◽

Polymerase Iii

The Epstein-Barr virus-encoded small RNA (EBER) genes are transcribed by RNA polymerase III, but their transcription unit appears to contain both class II and class III promoter elements. One of these promoter element, a TATA-like box which we call the EBER TATA box, or ETAB, is located in a position typical for a class II TATA box but contains G/C residues in the normal T/A motif and a conserved thymidine doublet. Experiments using chloramphenicol acetyltransferase constructs and mutations in the TATA box of the adenovirus major late promoter showed that the ETAB promoter element does not substitute for a class II TATA box. However, when the ETAB promoter element sequence was changed to a class II TATA box consensus sequence, the EBER 2 gene was transcribed in vitro by both RNA polymerases II and III. From these results, we conclude that the ETAB promoter element is important for the exclusive transcription of the EBER 2 gene by RNA polymerase III.

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TFIIIC1 acts through a downstream region to stabilize TFIIIC2 binding to RNA polymerase III promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6841 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6841-6850 ◽

Cited By ~ 38

Author(s):

Z Wang ◽

R G Roeder

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Weak Interaction ◽

Strong Interaction ◽

Rna Polymerase Iii ◽

Cooperative Interaction ◽

In Vitro System ◽

Polymerase Iii ◽

Footprint Analysis

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.

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A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system

Gene ◽

10.1016/j.gene.2006.10.011 ◽

2007 ◽

Vol 392 (1-2) ◽

pp. 7-13 ◽

Cited By ~ 6

Author(s):

Yasushi Yukawa ◽

Takaharu Mizutani ◽

Kazuhito Akama ◽

Masahiro Sugiura

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

In Vitro Transcription ◽

Trna Genes ◽

Transcription System ◽

Polymerase Iii ◽

Chromosome I

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Cloning and Characterization of Two Evolutionarily Conserved Subunits (TFIIIC102 and TFIIIC63) of Human TFIIIC and Their Involvement in Functional Interactions with TFIIIB and RNA Polymerase III

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4944 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4944-4952 ◽

Cited By ~ 39

Author(s):

Yng-Ju Hsieh ◽

Zhengxin Wang ◽

Robert Kovelman ◽

Robert G. Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Iii ◽

Structure And Function ◽

Class Iii ◽

Tetratricopeptide Repeats ◽

Helix Loop Helix ◽

Polymerase Iii ◽

And Function

ABSTRACT Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that mediates transcription of class III genes through direct recognition of promoters (for tRNA and virus-associated RNA genes) or promoter-TFIIIA complexes (for the 5S RNA gene) and subsequent recruitment of TFIIIB and RNA polymerase III. We describe the cognate cDNA cloning and characterization of two subunits (hTFIIIC63 and hTFIIIC102) that are present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC and are related in structure and function to two yeast TFIIIC subunits (yTFIIIC95 and yTFIIIC131) previously shown to interact, respectively, with the promoter (A box) and with a subunit of yeast TFIIIB. hTFIIIC63 and hTFIIIC102 show parallel in vitro interactions with the homologous human TFIIIB and RNA polymerase III components, as well as additional interactions that may facilitate both TFIIIB and RNA polymerase III recruitment. These include novel interactions of hTFIIIC63 with hTFIIIC102, with hTFIIIB90, and with hRPC62, in addition to the hTFIIIC102–hTFIIIB90 and hTFIIIB90–hRPC39 interactions that parallel the previously described interactions in yeast. As reported for yTFIIIC131, hTFIIIC102 contains acidic and basic regions, tetratricopeptide repeats (TPRs), and a helix-loop-helix domain, and mutagenesis studies have implicated the TPRs in interactions both with hTFIIIC63 and with hTFIIIB90. These observations further document conservation from yeast to human of the structure and function of the RNA polymerase III transcription machinery, but in addition, they provide new insights into the function of hTFIIIC and suggest direct involvement in recruitment of both TFIIIB and RNA polymerase III.

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CK2 Forms a Stable Complex with TFIIIB and Activates RNA Polymerase III Transcription in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3757-3768.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3757-3768 ◽

Cited By ~ 53

Author(s):

Imogen M. Johnston ◽

Simon J. Allison ◽

Jennifer P. Morton ◽

Laura Schramm ◽

Pamela H. Scott ◽

...

Keyword(s):

Rna Polymerase ◽

Mammalian Cells ◽

Rna Polymerase Iii ◽

Stable Complex ◽

Class Iii ◽

Potent Effect ◽

Cell Extracts ◽

Polymerase Iii ◽

Chemical Inhibitors

ABSTRACT CK2 is a highly conserved protein kinase with growth-promoting and oncogenic properties. It is known to activate RNA polymerase III (PolIII) transcription in Saccharomyces cerevisiae and is shown here to also exert a potent effect on PolIII in mammalian cells. Peptide and chemical inhibitors of CK2 block PolIII transcription in human cell extracts. Furthermore, PolIII transcription in mammalian fibroblasts is decreased significantly when CK2 activity is compromised by chemical inhibitors, antisense oligonucleotides, or kinase-inactive mutants. Coimmunoprecipitation and cofractionation show that endogenous human CK2 associates stably and specifically with the TATA-binding protein-containing factor TFIIIB, which brings PolIII to the initiation site of all class III genes. Serum stimulates TFIIIB phosphorylation in vivo, an effect that is diminished by inhibitors of CK2. Binding to TFIIIC2 recruits TFIIIB to most PolIII promoters; this interaction is compromised specifically by CK2 inhibitors. The data suggest that CK2 stimulates PolIII transcription by binding and phosphorylating TFIIIB and facilitating its recruitment by TFIIIC2. CK2 also activates PolI transcription in mammals and may therefore provide a mechanism to coregulate the output of PolI and PolIII. CK2 provides a rare example of an endogenous activity that operates on the PolIII system in both mammals and yeasts. Such evolutionary conservation suggests that this control may be of fundamental importance.

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TFIIIR is an isoleucine tRNA

Molecular and Cellular Biology ◽

10.1128/mcb.14.6.3588-3595.1994 ◽

1994 ◽

Vol 14 (6) ◽

pp. 3588-3595

Author(s):

H M Dunstan ◽

L S Young ◽

K U Sprague

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Silk Gland ◽

Class Iii ◽

Nuclear Extracts ◽

Hybrid Selection ◽

Class Iii Genes ◽

Selection Of

Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).

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Induction of specific transcription by RNA polymerase III in transformed cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3068-3076.1986 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3068-3076

Author(s):

M F Carey ◽

K Singh ◽

M Botchan ◽

N R Cozzarelli

Keyword(s):

Rna Polymerase ◽

Gene Family ◽

Rna Polymerase Iii ◽

Simian Virus 40 ◽

In Vitro Transcription ◽

Simian Virus ◽

Intrinsic Signals ◽

Polymerase Iii ◽

Pol Iii

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

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Analysis of transcription factors binding to the human 7SL RNA gene promoter

Biochemistry and Cell Biology ◽

10.1139/o99-051 ◽

1999 ◽

Vol 77 (5) ◽

pp. 431-438 ◽

Cited By ~ 8

Author(s):

Jürgen Müller ◽

Bernd-Joachim Benecke

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Gene Promoter ◽

7Sl Rna ◽

Polymerase Iii ◽

Responsive Element

Transcription of the human 7SL RNA gene by RNA polymerase III depends on the concerted action of transcription factors binding to the gene-internal and gene-external parts of its promoter. Here, we investigated which transcription factors interact with the human 7SL RNA gene promoter and which are required for transcription of the human 7SL RNA gene. A-box/B-box elements were previously identified in 5S RNA, tRNA, and virus associated RNA genes and are recognized by transcription factor IIIC (TFIIIC). The gene-internal promoter region of the human 7SL RNA gene shows only limited similarity to those elements. Nevertheless, competition experiments and the use of highly enriched factor preparations demonstrate that TFIIIC is required for human 7SL transcription. The gene-external part of the promoter includes an authentic cAMP-responsive element previously identified in various RNA polymerase II promoters. Here we demonstrate that members of the activating transcription factor/cyclic AMP-responsive element binding protein (ATF/CREB) transcription factor family bind specifically to this element in vitro. However, the human 7SL RNA gene is not regulated by cAMP in vivo. Furthermore, in vitro transcription of the gene does not depend on ATF/CREB transcription factors. It rather appears that a transcription factor with DNA-binding characteristics like ATF/CREB proteins but otherwise different properties is required for human 7SL RNA transcription.Key words: 7SL RNA, ATF, CRE, TFIIIC, RNA polymerase III.

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Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions.

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3117 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3117-3127 ◽

Cited By ~ 25

Author(s):

M S Kasher ◽

D Pintel ◽

D C Ward

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Specific Activity ◽

Rna Polymerase Iii ◽

Adenovirus Type ◽

Stable Complex ◽

Polymerase Iii ◽

S100 Extract ◽

Affinity Resin ◽

A Site

Plasmid DNA containing the adenovirus type 2 genes for VA RNA was linearized at a site distal to the gene, end labeled with a biotin-nucleotide analog of TTP, and incubated with avidin to form an avidin-biotinylated DNA complex. HeLa cell S100 extracts containing crude RNA polymerase III and transcription factors (TFs) IIIB and IIIC were programmed with the avidin-biotin-VA DNA to allow stable complex formation (A.B. Lassar, P.L. Martin, and R.G. Roeder, Science 222:740-748, 1983). Chromatography of the programmed extract over a biotin-cellulose affinity resin resulted in the selective, and virtually quantitative, retention of one of two stable preinitiation complexes, either VA-IIIC or VA-IIIC-IIIB, depending on the length of template incubation in the S100 extract. After washing the resin with 0.10 M and 0.25 M KCl to remove RNA polymerase III and nonspecifically bound proteins, respectively, TFIIIC was eluted from the VA-IIIC complex by the addition of 1.5 M KCl. The VA-IIIC-IIIB complex exhibited a higher salt stability. Most of TFIIIB and some TFIIIC were released by the addition of 1.5 M KCl; however, the majority of TFIIIC activity was recovered only after a subsequent 3.0 M KCl elution. The specific activity of the TFIIIC in the 3.0 M KCl fraction was 770-fold higher than that in the S100 extract, while the protein content of the 1.5 and 3.0 M KCl fractions was reduced 7,500- and 100,000-fold, respectively.

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