scholarly journals Structure of the human RNA polymerase I elongation complex

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Dan Zhao ◽  
Weida Liu ◽  
Ke Chen ◽  
Zihan Wu ◽  
Huirong Yang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Elongation Complex ◽  
Mismatched Dna ◽  
Pol I ◽  
Complex Organization ◽  
Polymerase I ◽  
Zinc Ribbon ◽  
Cellular Transcription ◽  
Single Subunit

AbstractEukaryotic RNA polymerase I (Pol I) transcribes ribosomal DNA and generates RNA for ribosome synthesis. Pol I accounts for the majority of cellular transcription activity and dysregulation of Pol I transcription leads to cancers and ribosomopathies. Despite extensive structural studies of yeast Pol I, structure of human Pol I remains unsolved. Here we determined the structures of the human Pol I in the pre-translocation, post-translocation, and backtracked states at near-atomic resolution. The single-subunit peripheral stalk lacks contacts with the DNA-binding clamp and is more flexible than the two-subunit stalk in yeast Pol I. Compared to yeast Pol I, human Pol I possesses a more closed clamp, which makes more contacts with DNA. The Pol I structure in the post-cleavage backtracked state shows that the C-terminal zinc ribbon of RPA12 inserts into an open funnel and facilitates “dinucleotide cleavage” on mismatched DNA–RNA hybrid. Critical disease-associated mutations are mapped on Pol I regions that are involved in catalysis and complex organization. In summary, the structures provide new sights into human Pol I complex organization and efficient proofreading.

scholarly journals Structure of the human RNA Polymerase I Elongation Complex

2021 ◽  
Author(s):  
Yanhui Xu ◽  
Dan Zhao ◽  
Weida Liu ◽  
Ke Chen ◽  
Zihan Wu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Human Cells ◽  
Rna Polymerase I ◽  
Elongation Complex ◽  
Mismatched Dna ◽  
Pol I ◽  
Complex Organization ◽  
Polymerase I ◽  
Zinc Ribbon

Abstract Eukaryotic RNA polymerase I (Pol I) transcribes ribosomal DNA and generates RNA for ribosome synthesis. Pol I accounts for the majority of cellular transcription activity and dysregulation of Pol I transcription leads to cancers and ribosomopathies. Despite extensive structural studies of yeast Pol I, structure of human Pol I remains unsolved. Here, we determined the structures of the human Pol I in the pre-translocation, post-translocation, and backtracked states at near-atomic resolution. The single-subunit peripheral stalk lacks contacts with the DNA-binding clamp and is more flexible than the two-subunit stalk in yeast Pol I. Compared to yeast Pol I, human Pol I possesses a more closed clamp, which makes more contacts with DNA and may support more efficient transcription in human cells. The Pol I structure in the post-cleavage backtracked state shows that the C-terminal zinc ribbon of RPA12 inserts into an open funnel and facilitates “dinucleotide cleavage” on mismatched DNA-RNA hybrid. Critical disease-associated mutations are mapped on Pol I regions that are involved in catalysis and complex organization. In summary, the structures provide new sights into human Pol I complex organization and efficient proofreading, consistent with requirement of efficient transcription of ribosomal DNA in human cells.

scholarly journals Structural basis of RNA polymerase I stalling at UV light-induced DNA damage

2018 ◽  
Vol 115 (36) ◽  
pp. 8972-8977 ◽  
Author(s):  
Marta Sanz-Murillo ◽  
Jun Xu ◽  
Georgiy A. Belogurov ◽  
Olga Calvo ◽  
David Gil-Carton ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Uv Light ◽  
Rna Polymerase I ◽  
Pyrimidine Dimer ◽  
Structural Basis ◽  
Elongation Complex ◽  
Pol I ◽  
Polymerase I

RNA polymerase I (Pol I) transcribes ribosomal DNA (rDNA) to produce the ribosomal RNA (rRNA) precursor, which accounts for up to 60% of the total transcriptional activity in growing cells. Pol I monitors rDNA integrity and influences cell survival, but little is known about how this enzyme processes UV-induced lesions. We report the electron cryomicroscopy structure of Pol I in an elongation complex containing a cyclobutane pyrimidine dimer (CPD) at a resolution of 3.6 Å. The structure shows that the lesion induces an early translocation intermediate exhibiting unique features. The bridge helix residue Arg1015 plays a major role in CPD-induced Pol I stalling, as confirmed by mutational analysis. These results, together with biochemical data presented here, reveal the molecular mechanism of Pol I stalling by CPD lesions, which is distinct from Pol II arrest by CPD lesions. Our findings open the avenue to unravel the molecular mechanisms underlying cell endurance to lesions on rDNA.

scholarly journals Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription

2010 ◽  
Vol 84 (11) ◽  
pp. 5824-5835 ◽  
Author(s):  
Takahiro Masaki ◽  
Ryosuke Suzuki ◽  
Mohsan Saeed ◽  
Ken-ichi Mori ◽  
Mami Matsuda ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Cell Line ◽  
Viral Genome ◽  
Rna Polymerase I ◽  
Virion Assembly ◽  
Pol I ◽  
Positive Strand Rna ◽  
Polymerase I

ABSTRACT In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up- and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.

scholarly journals RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

2020 ◽  
Vol 295 (15) ◽  
pp. 4782-4795 ◽  
Author(s):  
Philipp E. Merkl ◽  
Michael Pilsl ◽  
Tobias Fremter ◽  
Katrin Schwank ◽  
Christoph Engel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Dna Templates ◽  
Pol Ii ◽  
Naked Dna ◽  
Pol I ◽  
Intrinsic Capacity ◽  
Pol Iii ◽  
Polymerase I

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

scholarly journals Molecular Topology of RNA Polymerase I Upstream Activation Factor

2020 ◽  
Vol 40 (13) ◽  
Author(s):  
Bruce A. Knutson ◽  
Marissa L. Smith ◽  
Alana E. Belkevich ◽  
Aula M. Fakhouri
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Dual Function ◽  
Molecular Topology ◽  
Content Type ◽  
Histone Fold ◽  
Topological Features ◽  
Pol I ◽  
Activation Factor ◽  
Polymerase I

ABSTRACT Upstream activation factor (UAF) is a multifunctional transcription factor in Saccharomyces cerevisiae that plays dual roles in activating RNA polymerase I (Pol I) transcription and repression of Pol II. For Pol I, UAF binds to a specific upstream element in the ribosomal DNA (rDNA) promoter and interacts with two other Pol I initiation factors, the TATA-binding protein (TBP) and core factor (CF). We used an integrated combination of chemical cross-linking mass spectrometry (CXMS), molecular genetics, protein biochemistry, and structural modeling to understand the topological framework responsible for UAF complex formation. Here, we report the molecular topology of the UAF complex, describe new structural and functional domains that play roles in UAF complex integrity, assembly, and biological function, and provide roles for previously identified UAF domains that include the Rrn5 SANT and histone fold domains. We highlight the role of new domains in Uaf30 that include an N-terminal winged helix domain and a disordered tethering domain as well as a BORCS6-like domain found in Rrn9. Together, our results reveal a unique network of topological features that coalesce around a histone tetramer-like core to form the dual-function UAF complex.

scholarly journals Role of TATA Binding Protein (TBP) in Yeast Ribosomal DNA Transcription by RNA Polymerase I: Defects in the Dual Functions of Transcription Factor UAF Cannot Be Suppressed by TBP

2001 ◽  
Vol 21 (7) ◽  
pp. 2292-2297 ◽  
Author(s):  
Imran Siddiqi ◽  
John Keener ◽  
Loan Vu ◽  
Masayasu Nomura
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Rrna Transcription ◽  
Pol I ◽  
Mutant Strains ◽  
Polymerase I

ABSTRACT Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269–5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5,RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF componentRRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

scholarly journals The RNA polymerase I transcription machinery

2006 ◽  
Vol 73 ◽  
pp. 203-216 ◽  
Author(s):  
Jackie Russell ◽  
Joost C.B.M. Zomerdijk
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Growth Conditions ◽  
Rna Polymerase I ◽  
Cellular Growth ◽  
Rrna Synthesis ◽  
Transcription Machinery ◽  
Pol I ◽  
A Cell ◽  
Polymerase I

The rRNAs constitute the catalytic and structural components of the ribosome, the protein synthesis machinery of cells. The level of rRNA synthesis, mediated by Pol I (RNA polymerase I), therefore has a major impact on the life and destiny of a cell. In order to elucidate how cells achieve the stringent control of Pol I transcription, matching the supply of rRNA to demand under different cellular growth conditions, it is essential to understand the components and mechanics of the Pol I transcription machinery. In this review, we discuss: (i) the molecular composition and functions of the Pol I enzyme complex and the two main Pol I transcription factors, SL1 (selectivity factor 1) and UBF (upstream binding factor); (ii) the interplay between these factors during pre-initiation complex formation at the rDNA promoter in mammalian cells; and (iii) the cellular control of the Pol I transcription machinery.

scholarly journals Modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of RNA polymerase I transcription

2005 ◽  
Vol 86 (8) ◽  
pp. 2315-2322 ◽  
Author(s):  
Rajeev Banerjee ◽  
Mary K. Weidman ◽  
Sonia Navarro ◽  
Lucio Comai ◽  
Asim Dasgupta
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Selectivity Factor ◽  
Pol I ◽  
Binding Factor ◽  
Infected Cells ◽  
Upstream Binding Factor ◽  
Polymerase I

Soon after infection, poliovirus (PV) shuts off host-cell transcription, which is catalysed by all three cellular RNA polymerases. rRNA constitutes more than 50 % of all cellular RNA and is transcribed from rDNA by RNA polymerase I (pol I). Here, evidence has been provided suggesting that both pol I transcription factors, SL-1 (selectivity factor) and UBF (upstream binding factor), are modified and inactivated in PV-infected cells. The viral protease 3Cpro appeared to cleave the TATA-binding protein-associated factor 110 (TAF110), a subunit of the SL-1 complex, into four fragments in vitro. In vitro protease-cleavage assays using various mutants of TAF110 and purified 3Cpro indicated that the Q265G266 and Q805G806 sites were cleaved by 3Cpro. Both SL-1 and UBF were depleted in PV-infected cells and their disappearance correlated with pol I transcription inhibition. rRNA synthesis from a template containing a human pol I promoter demonstrated that both SL-1 and UBF were necessary to restore pol I transcription fully in PV-infected cell extracts. These results suggested that both SL-1 and UBF are transcriptionally inactivated in PV-infected HeLa cells.

scholarly journals Histone Acetyltransferase and Protein Kinase Activities Copurify with a Putative Xenopus RNA Polymerase I Holoenzyme Self-Sufficient for Promoter-Dependent Transcription

1999 ◽  
Vol 19 (1) ◽  
pp. 796-806 ◽  
Author(s):  
Annie-Claude Albert ◽  
Michael Denton ◽  
Milko Kermekchiev ◽  
Craig S. Pikaard
Keyword(s):  
Rna Polymerase ◽  
Histone Acetyltransferase ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Large Mass ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Pol I ◽  
Coomassie Blue ◽  
Polymerase I

ABSTRACT Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of ∼2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.

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