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

2002 ◽  
Vol 22 (11) ◽  
pp. 3757-3768 ◽  
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.

scholarly journals Maf1p, a Negative Effector of RNA Polymerase III inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (15) ◽  
pp. 5031-5040 ◽  
Author(s):  
Krzysztof Pluta ◽  
Olivier Lefebvre ◽  
Nancy C. Martin ◽  
Wieslaw J. Smagowicz ◽  
David R. Stanford ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Growth Defect ◽  
Class Iii ◽  
Gene Encoding ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Although yeast RNA polymerase III (Pol III) and the auxiliary factors TFIIIC and TFIIIB are well characterized, the mechanisms of class III gene regulation are poorly understood. Previous studies identified MAF1, a gene that affects tRNA suppressor efficiency and interacts genetically with Pol III. We show here that tRNA levels are elevated in maf1 mutant cells. In keeping with the higher levels of tRNA observed in vivo, the in vitro rate of Pol III RNA synthesis is significantly increased in maf1cell extracts. Mutations in the RPC160 gene encoding the largest subunit of Pol III which reduce tRNA levels were identified as suppressors of the maf1 growth defect. Interestingly, Maf1p is located in the nucleus and coimmunopurifies with epitope-tagged RNA Pol III. These results indicate that Maf1p acts as a negative effector of Pol III synthesis. This potential regulator of Pol III transcription is likely conserved since orthologs of Maf1p are present in other eukaryotes, including humans.

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

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45 ◽  
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

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.

scholarly journals RNA Polymerase III Transcription Factor IIIB Is a Target for Repression by Pocket Proteins p107 and p130

1999 ◽  
Vol 19 (6) ◽  
pp. 4255-4261 ◽  
Author(s):  
Josephine E. Sutcliffe ◽  
Carol A. Cairns ◽  
Angela McLees ◽  
Simon J. Allison ◽  
Kerrie Tosh ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Interaction ◽  
Double Knockout ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Pol Iii ◽  
Related Proteins

ABSTRACT RNA polymerase III (Pol III) transcription is subject to repression by the retinoblastoma protein RB, both in vitro and in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88–90, 1996). This is achieved through a direct interaction between RB and TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061–2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755–14761, 1997). p107 and p130 are two closely related proteins that display 30 to 35% identity with the RB polypeptide and share some of its functions. We show that p107 and p130 can both repress Pol III transcription in transient transfection assays or when added to cell extracts. Pull-down assays and immunoprecipitations using recombinant components demonstrate that a subunit of TFIIIB interacts physically with p107 and p130. In addition, endogenous TFIIIB is shown by cofractionation and coimmunoprecipitation to associate stably with both p107 and p130. Disruption of this interaction in vivo by using the E7 oncoprotein of human papillomavirus results in a marked increase in Pol III transcription. Pol III activity is also deregulated in fibroblasts derived from p107 p130 double knockout mice. We conclude that TFIIIB is targeted for repression not only by RB but also by its relatives p107 and p130.

scholarly journals Functions of paralogous RNA polymerase III subunits POLR3G and POLR3GL in mouse development

2020 ◽  
Vol 117 (27) ◽  
pp. 15702-15711
Author(s):  
Xiaoling Wang ◽  
Alan Gerber ◽  
Wei-Yi Chen ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Knockout Mice ◽  
Mammalian Cells ◽  
Target Genes ◽  
Rna Polymerase Iii ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Polymerase Iii ◽  
Pol Iii

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIβ). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.

A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III

1994 ◽  
Vol 14 (1) ◽  
pp. 822-830
Author(s):  
G Rameau ◽  
K Puglia ◽  
A Crowe ◽  
I Sethy ◽  
I Willis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structural Motif ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Polymerase Iii ◽  
Rate Limiting

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

Stable transcription complex on a class III gene in a minichromosome

1985 ◽  
Vol 5 (1) ◽  
pp. 40-45
Author(s):  
A B Lassar ◽  
D H Hamer ◽  
R G Roeder
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Stable Complex ◽  
Trna Genes ◽  
Class Iii ◽  
5S Rna ◽  
Polymerase Iii

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.

scholarly journals A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 822-830 ◽  
Author(s):  
G Rameau ◽  
K Puglia ◽  
A Crowe ◽  
I Sethy ◽  
I Willis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structural Motif ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Polymerase Iii ◽  
Rate Limiting

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

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

1993 ◽  
Vol 13 (5) ◽  
pp. 2655-2665 ◽  
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.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

scholarly journals Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Alexis V. Stutzman ◽  
April S. Liang ◽  
Vera Beilinson ◽  
Kohta Ikegami
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Mammalian Cells ◽  
Sex Chromosome ◽  
Rna Polymerase Iii ◽  
Chromatin Organization ◽  
Control Of Gene Expression ◽  
C Elegans ◽  
Polymerase Iii ◽  
The Individual

Abstract Background Chromatin organization is central to precise control of gene expression. In various eukaryotic species, domains of pervasive cis-chromatin interactions demarcate functional domains of the genomes. In nematode Caenorhabditis elegans, however, pervasive chromatin contact domains are limited to the dosage-compensated sex chromosome, leaving the principle of C. elegans chromatin organization unclear. Transcription factor III C (TFIIIC) is a basal transcription factor complex for RNA polymerase III, and is implicated in chromatin organization. TFIIIC binding without RNA polymerase III co-occupancy, referred to as extra-TFIIIC binding, has been implicated in insulating active and inactive chromatin domains in yeasts, flies, and mammalian cells. Whether extra-TFIIIC sites are present and contribute to chromatin organization in C. elegans remains unknown. Results We identified 504 TFIIIC-bound sites absent of RNA polymerase III and TATA-binding protein co-occupancy characteristic of extra-TFIIIC sites in C. elegans embryos. Extra-TFIIIC sites constituted half of all identified TFIIIC binding sites in the genome. Extra-TFIIIC sites formed dense clusters in cis. The clusters of extra-TFIIIC sites were highly over-represented within the distal arm domains of the autosomes that presented a high level of heterochromatin-associated histone H3K9 trimethylation (H3K9me3). Furthermore, extra-TFIIIC clusters were embedded in the lamina-associated domains. Despite the heterochromatin environment of extra-TFIIIC sites, the individual clusters of extra-TFIIIC sites were devoid of and resided near the individual H3K9me3-marked regions. Conclusion Clusters of extra-TFIIIC sites were pervasive in the arm domains of C. elegans autosomes, near the outer boundaries of H3K9me3-marked regions. Given the reported activity of extra-TFIIIC sites in heterochromatin insulation in yeasts, our observation raised the possibility that TFIIIC may also demarcate heterochromatin in C. elegans.

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