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.

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 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.

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 Structural basis of RNA polymerase III transcription repression by Maf1

2019 ◽  
Author(s):  
Matthias K. Vorländer ◽  
Florence Baudin ◽  
Robyn D. Moir ◽  
René Wetzel ◽  
Wim J. H. Hagen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Structural Basis ◽  
Metabolic Efficiency ◽  
Transcription Repression ◽  
Polymerase Iii ◽  
Wide Range ◽  
Pol Iii

ABSTRACTMaf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

scholarly journals TATA-Like Boxes in RNA Polymerase III Promoters: Requirements for Nucleotide Sequences

2020 ◽  
Vol 21 (10) ◽  
pp. 3706 ◽  
Author(s):  
Karina A. Tatosyan ◽  
Danil V. Stasenko ◽  
Anastasia P. Koval ◽  
Irina K. Gogolevskaya ◽  
Dmitri A. Kramerov
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Nucleotide Substitutions ◽  
Short Interspersed Elements ◽  
Polymerase Iii ◽  
Transcription Efficiency ◽  
Flanking Sequences ◽  
Pol Iii ◽  
Rna Genes

tRNA and some other non-coding RNA genes are transcribed by RNA polymerase III (pol III), due to the presence of intragenic promoter, consisting of boxes A and B spaced by 30–40 bp. Such pol III promoters, called type 2, are also intrinsic to Short Interspersed Elements (SINEs). The contribution of 5′-flanking sequences to the transcription efficiency of genes containing type 2 promoters is still studied insufficiently. Here, we studied this issue, focusing on the genes of two small non-coding RNAs (4.5SH and 4.5SI), as well as B1 and B2 SINEs from the mouse genome. We found that the regions from position −31 to −24 may significantly influence the transcription of genes and SINEs. We studied the influence of nucleotide substitutions in these sites, representing TATA-like boxes, on transcription of 4.5SH and 4.5SI RNA genes. As a rule, the substitutions of A and T to G or C reduced the transcription level, although the replacement of C with A also lowered it. In 4.5SH gene, five distal nucleotides of −31/−24 box (TTCAAGTA) appeared to be the most important, while in the box −31/−24 of 4.5SI gene (CTACATGA), all nucleotides, except for the first one, contributed significantly to the transcription efficiency. Random sequences occurring at positions −31/−24 upstream of SINE copies integrated into genome, promoted their transcription with different efficacy. In the 5′-flanking sequences of 4.5SH and 4.5SI RNA genes, the recognition sites of CREB, C/EBP, and Sp1 factors were found, and their deletion decreased the transcription.

scholarly journals 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 RNA polymerase III transcription in synthetic nuclei assembled in vitro from defined DNA templates.

1995 ◽  
Vol 15 (9) ◽  
pp. 4873-4883 ◽  
Author(s):  
K S Ullman ◽  
D J Forbes
Keyword(s):  
Rna Polymerase ◽  
Nuclear Structure ◽  
De Novo ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Dna Encoding ◽  
Nuclear Assembly ◽  
Polymerase Iii ◽  
Pol Iii

Although much is known of the basic control of transcription, little is understood of the way in which the structural organization of the nucleus affects transcription. Synthetic nuclei, assembled de novo in extracts of Xenopus eggs, would be predicted to have a large potential for approaching the role of nuclear structure in RNA biogenesis. Synthetic nuclei provide a system in which the genetic content of the nuclei, as well as the structural and enzymatic proteins within the nuclei, can be manipulated. In this study, we have begun to examine transcription in such nuclei by using the most simple of templates, RNA polymerase III (pol III)-transcribed genes. DNA encoding tRNA or 5S genes was added to an assembly extract, and nuclei were formed entirely from the pol III templates. Conditions which allowed nuclear assembly and pol III transcription to take place efficiently and simultaneously in the assembly extract were found. To examine whether pol III transcription could initiate within synthetic nuclei, or instead was inhibited in nuclei and initiated only on rare unincorporated templates, we identified transcriptional inhibitors that were excluded from nuclei. We found that these inhibitors, heparin and dextran sulfate, blocked pol III transcription in the absence of assembly but did not do so following nuclear assembly. At the concentrations used, the inhibitors had no deleterious effect on nuclear structure itself or on nuclear import. We conclude that pol III transcription is active in synthetic nuclei, and this conclusion is further strengthened by the finding that pol III transcripts could be coisolated with synthetic nuclei. The rapid and direct transcriptional analysis possible with pol III templates, coupled with the simple experimental criteria developed in this study for distinguishing between nuclear and non-nuclear transcription, should now allow a molecular analysis of the effect of nuclear structure on transcriptional and posttranscriptional control.

scholarly journals Essential Roles of Bdp1, a Subunit of RNA Polymerase III Initiation Factor TFIIIB, in Transcription and tRNA Processing

2002 ◽  
Vol 22 (10) ◽  
pp. 3264-3275 ◽  
Author(s):  
Akira Ishiguro ◽  
George A. Kassavetis ◽  
E. Peter Geiduschek
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Genomic Library ◽  
Rna Polymerase Iii ◽  
Rnase P ◽  
Initiation Factor ◽  
Physical Interaction ◽  
Polymerase Iii ◽  
A Subunit ◽  
Pol Iii

ABSTRACT The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 (τ131) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37°C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5′ ends. Maturation of tRNA was found to be aberrant in bdp1-Δ253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1Δ253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.

scholarly journals Mechanism of RNA polymerase III termination-associated reinitiation-recycling conferred by the essential function of the N terminal-and-linker domain of the C11 subunit

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Saurabh Mishra ◽  
Shaina H. Hasan ◽  
Rima M. Sakhawala ◽  
Shereen Chaudhry ◽  
Richard J. Maraia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Point Mutations ◽  
Cleavage Activity ◽  
Terminal Domain ◽  
Polymerase Iii ◽  
Essential Function ◽  
High Level ◽  
Pol Iii

AbstractRNA polymerase III achieves high level tRNA synthesis by termination-associated reinitiation-recycling that involves the essential C11 subunit and heterodimeric C37/53. The C11-CTD (C-terminal domain) promotes Pol III active center-intrinsic RNA 3′-cleavage although deciphering function for this activity has been complicated. We show that the isolated NTD (N-terminal domain) of C11 stimulates Pol III termination by C37/53 but not reinitiation-recycling which requires the NTD-linker (NTD-L). By an approach different from what led to current belief that RNA 3′-cleavage activity is essential, we show that NTD-L can provide the essential function of Saccharomyces cerevisiae C11 whereas classic point mutations that block cleavage, interfere with active site function and are toxic to growth. Biochemical and in vivo analysis including of the C11 invariant central linker led to a model for Pol III termination-associated reinitiation-recycling. The C11 NTD and CTD stimulate termination and RNA 3′-cleavage, respectively, whereas reinitiation-recycling activity unique to Pol III requires only the NTD-linker. RNA 3′-cleavage activity increases growth rate but is nonessential.

Induction of specific transcription by RNA polymerase III in transformed cells

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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