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.

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 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 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.

DNA Origami as a DNA Repair Nanosensor at the Single-Molecule Level

2013 ◽  
Vol 52 (30) ◽  
pp. 7747-7750 ◽  
Author(s):  
Maria Tintoré ◽  
Isaac Gállego ◽  
Brendan Manning ◽  
Ramon Eritja ◽  
Carme Fàbrega
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Dna Origami ◽  
Single Molecule Level

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 Novel Small-Molecule Inhibitors of RNA Polymerase III

2003 ◽  
Vol 2 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Liping Wu ◽  
Jing Pan ◽  
Vala Thoroddsen ◽  
Deborah R. Wysong ◽  
Ronald K. Blackman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Polymerase Iii ◽  
Wild Type ◽  
Biochemical Assays ◽  
Transcription In Vitro ◽  
Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

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