scholarly journals Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias K. Vorländer ◽  
Anna Jungblut ◽  
Kai Karius ◽  
Florence Baudin ◽  
Helga Grötsch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dual Function ◽  
Start Site ◽  
Transcription Start ◽  
Initiation Complex ◽  
Pol Ii ◽  
Negative Stain ◽  
Pol Iii

Abstract Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

scholarly journals Structure of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation

2020 ◽  
Author(s):  
Matthias K. Vorländer ◽  
Anna Jungblut ◽  
Kai Karius ◽  
Florence Baudin ◽  
Helga Grötsch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dual Function ◽  
Start Site ◽  
Transcription Start ◽  
Initiation Complex ◽  
Pol Ii ◽  
Negative Stain ◽  
Pol Iii

ABSTRACTTranscription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP with an unexpected location of Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

scholarly journals The hRPC62 subunit of human RNA polymerase III displays helicase activity

2019 ◽  
Vol 47 (19) ◽  
pp. 10313-10326 ◽  
Author(s):  
Leyla El Ayoubi ◽  
Hélène Dumay-Odelot ◽  
Aleksandar Chernev ◽  
Fanny Boissier ◽  
Lionel Minvielle-Sébastia ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Complex Formation ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
5S Rna ◽  
Initiation Complex ◽  
Pol Ii ◽  
Pol Iii

Abstract In Eukaryotes, tRNAs, 5S RNA and U6 RNA are transcribed by RNA polymerase (Pol) III. Human Pol III is composed of 17 subunits. Three specific Pol III subunits form a stable ternary subcomplex (RPC62-RPC39-RPC32α/β) being involved in pre-initiation complex formation. No paralogues for subunits of this subcomplex subunits have been found in Pols I or II, but hRPC62 was shown to be structurally related to the general Pol II transcription factor hTFIIEα. Here we show that these structural homologies extend to functional similarities. hRPC62 as well as hTFIIEα possess intrinsic ATP-dependent 3′-5′ DNA unwinding activity. The ATPase activities of both proteins are stimulated by single-stranded DNA. Moreover, the eWH domain of hTFIIEα can replace the first eWH (eWH1) domain of hRPC62 in ATPase and DNA unwinding assays. Our results identify intrinsic enzymatic activities in hRPC62 and hTFIIEα.

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 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 Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

1996 ◽  
Vol 16 (10) ◽  
pp. 5801-5810 ◽  
Author(s):  
G N Zecherle ◽  
S Whelen ◽  
B D Hall
Keyword(s):  
Rna Polymerase ◽  
Ternary Complex ◽  
Rna Polymerase Iii ◽  
Phenotypic Change ◽  
Start Site ◽  
Transcription Start ◽  
Polymerase Iii ◽  
Nascent Transcripts ◽  
Mutant Genes

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex.

scholarly journals Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

2021 ◽  
Author(s):  
Sarah E Dremel ◽  
Frances L Sivrich ◽  
Jessica M Tucker ◽  
Britt A Glaunsinger ◽  
Neal A DeLuca
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Viral Infection ◽  
Rna Polymerase Iii ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Simplex Virus ◽  
Pol Iii ◽  
Hsv 1

RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host tRNA with a specific codon bias were not targeted, rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection is known to mediate host transcriptional shut off and lead to a depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

scholarly journals Engineered mini H1 promoters with dedicated RNA Polymerase II or III activity

2020 ◽  
pp. jbc.RA120.015386
Author(s):  
Zongliang Gao ◽  
Yme Ubeles van der Velden ◽  
Minghui Fan ◽  
Cynthia Alyssa van der Linden ◽  
Monique Vink ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Polymerase Activity ◽  
Attractive Alternative ◽  
Pol Ii ◽  
Guide Rnas ◽  
Non Coding Rna ◽  
Pol Iii ◽  
Short Hairpin Rnas

RNA polymerase III promoters such as 7SK, U6 and H1 are widely used for the expression of small non-coding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a non-coding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting the Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly-used H1 promoter in terms of activity and small promoter size, but also concerning safety by exclusive expression of the desired therapeutic transcript (either Pol II or Pol III, but not both).

DNA origami-based single-molecule force spectroscopy unravels the molecular basis of RNA Polymerase III pre-initiation complex stability

2019 ◽  
Author(s):  
Kevin Kramm ◽  
Tim Schröder ◽  
Jerome Gouge ◽  
Andrés Manuel Vera ◽  
Florian B. Heiss ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Rna Polymerase Iii ◽  
Dna Origami ◽  
Initiation Complex ◽  
Single Molecule Level ◽  
Initiation Factors ◽  
Strict Requirement ◽  
Rnap Ii ◽  
Pol Iii

AbstractThe TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor compound the fundamental core of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional intiation factor Bdp1, which is unique to the RNA polymerase (RNAP) III sytem, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain, that arises from DNA compaction and transcriptional activity, on the efficiency of initiation complex formation. We made use of a new nanotechnological tool – a DNA origami-based force clamp - to follow the assembly of human initiation complexes in the Pol II and Pol III system at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is necessary and sufficient to stabilise TBP on a strained RNAP II promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of Pol III genes for the first time.

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.

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