Orientation and topography of RNA polymerase III in transcription complexes

1993 ◽  
Vol 13 (2) ◽  
pp. 942-952
Author(s):  
B Bartholomew ◽  
D Durkovich ◽  
G A Kassavetis ◽  
E P Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Leading Edge ◽  
Cross Linking ◽  
Transcription Bubble ◽  
Transcription Complexes ◽  
Pol Iii ◽  
Tyr Gene

A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor TFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B" components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.

scholarly journals Orientation and topography of RNA polymerase III in transcription complexes.

1993 ◽  
Vol 13 (2) ◽  
pp. 942-952 ◽  
Author(s):  
B Bartholomew ◽  
D Durkovich ◽  
G A Kassavetis ◽  
E P Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Leading Edge ◽  
Cross Linking ◽  
Transcription Bubble ◽  
Transcription Complexes ◽  
Pol Iii ◽  
Tyr Gene

A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor TFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B" components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.

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 A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

1997 ◽  
Vol 17 (10) ◽  
pp. 5823-5832 ◽  
Author(s):  
J L Goodier ◽  
H Fan ◽  
R J Maraia
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
Terminal Domain ◽  
La Protein ◽  
Pol Iii ◽  
Basic Region

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III 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 Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guillermo Abascal-Palacios ◽  
Laura Jochem ◽  
Carlos Pla-Prats ◽  
Fabienne Beuron ◽  
Alessandro Vannini
Keyword(s):  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Trna Gene ◽  
Integration Site ◽  
Rna Polymerase Iii ◽  
Structural Basis ◽  
Strand Transfer ◽  
Rna Pol Ii ◽  
A Subunit ◽  
Pol Iii

AbstractRetrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.

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 Hepatitis B virus X protein induces RNA polymerase III-dependent gene transcription and increases cellular TATA-binding protein by activating the Ras signaling pathway.

1997 ◽  
Vol 17 (12) ◽  
pp. 6838-6846 ◽  
Author(s):  
H D Wang ◽  
A Trivedi ◽  
D L Johnson
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Gene Transcription ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Tata Binding Protein ◽  
S2 Cells ◽  
B Virus ◽  
Pol Iii

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

scholarly journals A post-recruitment function for the RNA polymerase III transcription–initiation factor IIIB

1998 ◽  
Vol 95 (16) ◽  
pp. 9196-9201 ◽  
Author(s):  
George A. Kassavetis ◽  
Ashok Kumar ◽  
Garth A. Letts ◽  
E. Peter Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Rna Polymerase Iii ◽  
Active Role ◽  
Normal Function ◽  
Initiation Factor ◽  
Transcriptional Initiation ◽  
Pol Iii

Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B′′. Certain mutations in Saccharomyces cerevisiae Brf and B′′ retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB–DNA complexes bind pol III and position it appropriately over the transcriptional start site but do not form DNA strand-separated open promoter complexes. It is proposed that the normal function of TFIIIB combines pol III recruitment with an active role in a subsequent step of transcriptional initiation leading to promoter opening.

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