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Structure of human RNA polymerase III.

ABSTRACT: In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection. Here, we report a cryo-EM reconstruction at 4.0 Å of human Pol III, allowing mapping and rationalization of reported genetic mutations. Mutations causing neurodevelopmental defects cluster in hotspots affecting Pol III stability and/or biogenesis, whereas mutations affecting viral sensing are located in proximity to DNA binding regions, suggesting an impairment of Pol III cytosolic viral DNA-sensing. Integrating x-ray crystallography and SAXS, we also describe the structure of the higher eukaryote specific RPC5 C-terminal extension. Surprisingly, experiments in living cells highlight a role for this module in the assembly and stability of human Pol III.

SUBMITTER: Ramsay EP

PROVIDER: S-EPMC7747717 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structure of human RNA polymerase III elongation complex.

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.

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Characterization of human RNA polymerase III identifies orthologues for Saccharomyces cerevisiae RNA polymerase III subunits.

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.

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A structural perspective of human RNA polymerase III.

Project description:RNA polymerase III (Pol III) is a large multisubunit complex conserved in all eukaryotes that plays an essential role in producing a variety of short non-coding RNAs, such as tRNA, 5S rRNA and U6 snRNA transcripts. Pol III comprises of 17 subunits in both yeast and human with a 10-subunit core and seven peripheral subunits. Because of its size and complexity, Pol III has posed a formidable challenge to structural biologists. The first atomic cryogenic electron microscopy structure of yeast Pol III leading to the canonical view was reported in 2015. Within the last few years, the optimization of endogenous extract and purification procedure and the technical and methodological advances in cryogenic electron microscopy, together allow us to have a first look at the unprecedented details of human Pol III organization. Here, we look back on the structural studies of human Pol III and discuss them in the light of our current understanding of its role in eukaryotic transcription.

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Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells.

Project description:Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.

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Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters.

Project description:Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.

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Cryo-EM structures of human RNA Polymerase III

Project description: Not available

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The largest subunit of human RNA polymerase III is closely related to the largest subunit of yeast and trypanosome RNA polymerase III.

Project description:In both yeast and mammalian systems, considerable progress has been made toward the characterization of the transcription factors required for transcription by RNA polymerase III. However, whereas in yeast all of the RNA polymerase III subunits have been cloned, relatively little is known about the enzyme itself in higher eukaryotes. For example, no higher eukaryotic sequence corresponding to the largest RNA polymerase III subunit is available. Here we describe the isolation of cDNAs that encode the largest subunit of human RNA polymerase III, as suggested by the observations that (1) antibodies directed against the cloned protein immunoprecipitate an active enzyme whose sensitivity to different concentrations of alpha-amanitin is that expected for human RNA polymerase III; and (2) depletion of transcription extracts with the same antibodies results in inhibition of transcription from an RNA polymerase III, but not from an RNA polymerase II, promoter. Sequence comparisons reveal that regions conserved in the RNA polymerase I, II, and III largest subunits characterized so far are also conserved in the human RNA polymerase III sequence, and thus probably perform similar functions for the human RNA polymerase III enzyme.

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RNA polymerase III interactome

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

Structure of human mitochondrial RNA polymerase elongation complex.

Project description:Here we report the crystal structure of the human mitochondrial RNA polymerase (mtRNAP) transcription elongation complex, determined at 2.65-Å resolution. The structure reveals a 9-bp hybrid formed between the DNA template and the RNA transcript and one turn of DNA both upstream and downstream of the hybrid. Comparisons with the distantly related RNA polymerase (RNAP) from bacteriophage T7 indicates conserved mechanisms for substrate binding and nucleotide incorporation but also strong mechanistic differences. Whereas T7 RNAP refolds during the transition from initiation to elongation, mtRNAP adopts an intermediary conformation that is capable of elongation without refolding. The intercalating hairpin that melts DNA during T7 RNAP initiation separates RNA from DNA during mtRNAP elongation. Newly synthesized RNA exits toward the pentatricopeptide repeat (PPR) domain, a unique feature of mtRNAP with conserved RNA-recognition motifs.

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The hRPC62 subunit of human RNA polymerase III displays helicase activity.

Project description: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-RPC32?/?) 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 hTFIIE?. Here we show that these structural homologies extend to functional similarities. hRPC62 as well as hTFIIE? 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 hTFIIE? can replace the first eWH (eWH1) domain of hRPC62 in ATPase and DNA unwinding assays. Our results identify intrinsic enzymatic activities in hRPC62 and hTFIIE?.

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