BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth of Trypanosoma brucei

Parasitology ◽  
2015 ◽  
Vol 142 (13) ◽  
pp. 1563-1573 ◽  
Author(s):  
D. E. VÉLEZ-RAMÍREZ ◽  
L. E. FLORENCIO-MARTÍNEZ ◽  
G. ROMERO-MEZA ◽  
S. ROJAS-SÁNCHEZ ◽  
R. MORENO-CAMPOS ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Homology Modelling ◽  
Rna Polymerase Iii ◽  
Related Factor ◽  
Characteristic Structure ◽  
Rna Molecules ◽  
Polymerase Iii ◽  
Pol Iii

SUMMARYRNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

scholarly journals The transcription factor Sp1 modulates RNA polymerase III gene transcription by controlling BRF1 and GTF3C2 expression in human cells

2020 ◽  
Vol 295 (14) ◽  
pp. 4617-4630
Author(s):  
Feixia Peng ◽  
Ying Zhou ◽  
Juan Wang ◽  
Baoqiang Guo ◽  
Yun Wei ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Gene Transcription ◽  
Rna Polymerase Iii ◽  
Human Cells ◽  
Related Factor ◽  
Filamin A ◽  
Polymerase Iii ◽  
C Subunit ◽  
Pol Iii

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III–mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box–binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III–directed transcription and shed light on how Sp1 regulates cancer cell proliferation.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

scholarly journals Mechanism of Eukaryotic RNA Polymerase III Transcription Termination

Science ◽  
2013 ◽  
Vol 340 (6140) ◽  
pp. 1577-1580 ◽  
Author(s):  
Soren Nielsen ◽  
Yulia Yuzenkova ◽  
Nikolay Zenkin
Keyword(s):  
Rna Polymerase ◽  
Rna Secondary Structure ◽  
Common Ancestor ◽  
Rna Polymerase Iii ◽  
Rna Molecules ◽  
Polymerase Iii ◽  
Bacterial Rna ◽  
Catalytic Inactivation ◽  
The Common ◽  
Pol Iii

Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III–synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, which does not cause termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates Pol III release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multisubunit RNA polymerases.

scholarly journals High-Level Activation of Transcription of the Yeast U6 snRNA Gene in Chromatin by the Basal RNA Polymerase III Transcription Factor TFIIIC

2004 ◽  
Vol 24 (9) ◽  
pp. 3596-3606 ◽  
Author(s):  
Sushma Shivaswamy ◽  
George A. Kassavetis ◽  
Purnima Bhargava
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Role ◽  
U6 Snrna ◽  
Snrna Gene ◽  
Polymerase Iii ◽  
Activation Of Transcription ◽  
Pol Iii

ABSTRACT Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was ∼35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.

scholarly journals A Region of Bdp1 Necessary for Transcription Initiation That Is Located within the RNA Polymerase III Active Site Cleft

2015 ◽  
Vol 35 (16) ◽  
pp. 2831-2840 ◽  
Author(s):  
Hui-Lan Hu ◽  
Chih-Chien Wu ◽  
Jin-Cheng Lee ◽  
Hung-Ta Chen
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Active Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Terminal Region ◽  
Preinitiation Complex ◽  
Polymerase Iii ◽  
Active Site Cleft ◽  
Pol Iii

The RNA polymerase III (Pol III)-specific transcription factor Bdp1 is crucial to Pol III recruitment and promoter opening in transcription initiation, yet structural information is sparse. To examine its protein-binding targets within the preinitiation complex at the residue level, photoreactive amino acids were introduced intoSaccharomyces cerevisiaeBdp1. Mutations within the highly conserved SANT domain cross-linked to the transcription factor IIB (TFIIB)-related transcription factor Brf1, consistent with the findings of previous studies. In addition, we identified an essential N-terminal region that cross-linked with the Pol III catalytic subunit C128 as well as Brf1. Closer examination revealed that this region interacted with the C128 N-terminal region, the N-terminal half of Brf1, and the C-terminal domain of the C37 subunit, together positioning this region within the active site cleft of the preinitiation complex. With our functional data, our analyses identified an essential region of Bdp1 that is positioned within the active site cleft of Pol III and necessary for transcription initiation.

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.

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