RNA Polymerase III initially transcribing complex
Ontology highlight
ABSTRACT:
SUBMITTER: Matthias Vorländer
PROVIDER: EMPIAR-10190 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: Not available
| EMPIAR-10168 | biostudies-other
Project description:RNA polymerase III (Pol III) transcribes essential structured small RNAs, such as tRNAs, 5S rRNA and U6 snRNA. The transcriptional activity of Pol III is tightly controlled and its dysregulation is associated with human diseases, such as cancer. Human Pol III has two isoforms with difference only in one of its subunits RPC7 (α and β). Despite structural studies of yeast Pol III, structure of human Pol III remains unsolved. Here, we determined the structures of 17-subunit human Pol IIIα complex in the backtracked and post-translocation states, respectively. Human Pol III contains a generally conserved catalytic core, similar to that of yeast counterpart, and structurally unique RPC3-RPC6-RPC7 heterotrimer and RPC10. The N-ribbon of TFIIS-like RPC10 docks on the RPC4-RPC5 heterodimer and the C-ribbon inserts into the funnel of Pol III in the backtracked state but is more flexible in the post-translocation state. RPC7 threads through the heterotrimer and bridges the stalk and Pol III core module. The winged helix 1 domain of RPC6 and the N-terminal region of RPC7α stabilize each other and may prevent Maf1-mediated repression of Pol III activity. The C-terminal FeS cluster of RPC6 coordinates a network of interactions that mediate core-heterotrimer contacts and stabilize Pol III. Our structural analysis sheds new light on the molecular mechanism of human Pol IIIα-specific transcriptional regulation and provides explanations for upregulated Pol III activity in RPC7α-dominant cancer cells.
| S-EPMC8249397 | biostudies-literature
Project description:Extra TF(III)C (ETC) sites are chromosomal locations bound in vivo by the RNA polymerase III (Pol III) transcription factor III C (TF(III)C) complex, but are not necessarily associated with Pol III transcription. Although the location of ETC sequences are conserved in budding yeast, and similar sites are found in other organisms, their functions are largely unstudied. One such site, ETC6 in Saccharomyces cerevisiae, lies upstream of TFC6, a gene encoding a subunit of the TF(III)C complex itself. Promoter analysis shows that the ETC6 B-box sequence is involved in autoregulation of the TFC6 promoter. Mutation of ETC6 increases TFC6 mRNA levels, whereas mutation immediately upstream severely weakens promoter activity. A temperature-sensitive mutation in TFC3 that weakens DNA binding of TF(III)C also results in increased TFC6 mRNA levels; however, no increase is observed in mutants of TF(III)B or Pol III subunits, demonstrating a specific role for the TF(III)C complex in TFC6 promoter regulation. Chromatin immunoprecipitation shows an inverse relationship of TF(III)C occupancy at ETC6 versus TFC6 mRNA levels. Overexpression of TFC6 increases association of TF(III)C at ETC6 (and other loci) and results in reduced expression of a TFC6 promoter-URA3 reporter gene. Both of these effects are dependent on the ETC6 B-box. These results demonstrate that the TFC6 promoter is directly regulated by the TF(III)C complex, a demonstration of an RNA polymerase II promoter being directly responsive to a core Pol III transcription factor complex. This regulation could have implications in controlling global tRNA expression levels.
| S-EPMC3100981 | biostudies-literature
Project description:Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.
| S-EPMC5679246 | biostudies-literature
Project description:BackgroundAssembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as 'extra-transcriptional' effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites.ResultsGiven our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5'- and 3'-extended transcripts, and de-repression of adjacent genes and intergenic regions.ConclusionsThe results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome.
| S-EPMC4228148 | biostudies-literature
Project description:Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization.
| S-EPMC3511066 | biostudies-literature
Project description:Many regulatory proteins and complexes have been identified which influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding RNAs. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used proteomic approaches and pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to find identify the potential interactors through mass spectrometry-based proteomics. A large number of proteins were found in the interactome, which includes known chromatin modifiers, factors and regulators of transcription by pol I and pol II.
2019-01-02 | PXD003748 | Pride
Project description:Unlike Saccharomyces cerevisiae RNA polymerase III, human RNA polymerase III has not been entirely characterized. Orthologues of the yeast RNA polymerase III subunits C128 and C37 remain unidentified, and for many of the other subunits, the available information is limited to database sequences with various degrees of similarity to the yeast subunits. We have purified an RNA polymerase III complex and identified its components. We found that two RNA polymerase III subunits, referred to as RPC8 and RPC9, displayed sequence similarity to the RNA polymerase II RPB7 and RPB4 subunits, respectively. RPC8 and RPC9 associated with each other, paralleling the association of the RNA polymerase II subunits, and were thus paralogues of RPB7 and RPB4. Furthermore, the complex contained a prominent 80-kDa polypeptide, which we called RPC5 and which corresponded to the human orthologue of the yeast C37 subunit despite limited sequence similarity. RPC5 associated with RPC53, the human orthologue of S. cerevisiae C53, paralleling the association of the S. cerevisiae C37 and C53 subunits, and was required for transcription from the type 2 VAI and type 3 human U6 promoters. Our results provide a characterization of human RNA polymerase III and show that the RPC5 subunit is essential for transcription.
| S-EPMC134740 | biostudies-literature
Project description:Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated approximately 300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was alpha-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon alpha-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome.
| S-EPMC2082468 | biostudies-literature
Project description:In eukaryotes, RNA Polymerase III (Pol III) is specifically responsible for transcribing genes encoding tRNAs and other short non-coding RNAs. The recruitment of Pol III to tRNA-encoding genes requires the transcription factors (TF) IIIB and IIIC. TFIIIC has been described as a conserved, multi-subunit protein complex composed of two subcomplexes, called τA and τB. How these two subcomplexes are linked and how their interaction affects the formation of the Pol III pre-initiation complex (PIC) is poorly understood. Here we use chemical crosslinking mass spectrometry and determine the molecular architecture of TFIIIC. We further report the crystal structure of the essential TPR array from τA subunit τ131 and characterize its interaction with a central region of τB subunit τ138. The identified τ131-τ138 interacting region is essential in vivo and overlaps with TFIIIB-binding sites, revealing a crucial interaction platform for the regulation of tRNA transcription initiation.
| S-EPMC4490372 | biostudies-literature