scholarly journals Cell-cycle dependent organization and dynamics of RNA Polymerase I in live human cells

2017 ◽  
Author(s):  
William Conway ◽  
Won-Ki Cho ◽  
Namrata Jayanth ◽  
Susan Mullen ◽  
Ibrahim I Cissé
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Global Gene Expression ◽  
Human Cells ◽  
Rna Polymerase I ◽  
Temporal Organization ◽  
Pol I ◽  
Polymerase I

RNA Polymerase I (Pol I) is responsible for over 60% of transcriptional output in human cells, yet basic questions concerning the spatial and temporal organization of the polymerase remain unanswered. Here we investigate how mammalian cells rely on Pol I organization throughout the cell cycle to balance different needs, from complete transcription shut down to massive increase in protein synthesis (and thus ribosomal RNA synthesis) before cell division. In contrast to our previous reports on RNA Polymerase II, Pol I clusters are stable with active transcription, and the presence of transient Pol I clusters correlates with inactive ribosomal transcription. Our results suggest that both stable and transient populations Pol I clusters co-exist in individual living cells, and their relative fraction may directly reflect the global gene expression need of the cell.

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 RNA polymerase I transcription fidelity, speed and processivity depend on the interplay of its lobe binding subunits

2018 ◽  
Author(s):  
Philipp E. Merkl ◽  
Michael Pilsl ◽  
Tobias Fremter ◽  
Gernot Längst ◽  
Philipp Milkereit ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Transcription System ◽  
Pol I ◽  
Polymerase I ◽  
Rna Genes

AbstractEukaryotic RNA polymerases I and III (Pol I and III) consist of core subunits, which are conserved in RNA polymerase II (Pol II). Additionally, Pol I and III have specific subunits, associating with the so-called ‘lobe’ structure first described within Pol II. In Pol I of the yeast S. cerevisiae, these are Rpa34.5, and the N-terminal domains of Rpa49 and Rpa12.2, here referred to as the lobe-binding module (lb-module). We analyzed functions of the lb-module in a defined in vitro transcription system. Cooperation between lb-module components influenced transcription fidelity, elongation speed, and release of stalled Pol I complexes to continue elongation. Interestingly, lb-module containing Pol I and III, but not Pol II, were able to transcribe nucleosomal templates. Our data suggest, how the Pol I specific subunits may contribute to accurate and processive transcription of ribosomal RNA genes.

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.

Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I

1993 ◽  
Vol 13 (4) ◽  
pp. 2441-2455
Author(s):  
M Oakes ◽  
Y Nogi ◽  
M W Clark ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Structural Element ◽  
Nucleolar Structure ◽  
Pol I ◽  
Polymerase I

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

scholarly journals Structural alterations of the nucleolus in mutants of Saccharomyces cerevisiae defective in RNA polymerase I.

1993 ◽  
Vol 13 (4) ◽  
pp. 2441-2455 ◽  
Author(s):  
M Oakes ◽  
Y Nogi ◽  
M W Clark ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Structural Element ◽  
Nucleolar Structure ◽  
Pol I ◽  
Polymerase I

We have previously constructed mutants of Saccharomyces cerevisiae in which the gene for the second-largest subunit of RNA polymerase I (Pol I) is deleted. In these mutants, rRNA is synthesized by RNA polymerase II from a hybrid gene consisting of the 35S rRNA coding region fused to the GAL7 promoter on a plasmid. These strains thus grow in galactose but not glucose media. By immunofluorescence microscopy using antibodies against the known nucleolar proteins SSB1 and fibrillarin, we found that the intact crescent-shaped nucleolar structure is absent in these mutants; instead, several granules (called mininucleolar bodies [MNBs]) that stained with these antibodies were seen in the nucleus. Conversion of the intact nucleolar structure to MNBs was also observed in Pol I temperature-sensitive mutants at nonpermissive temperatures. These MNBs may structurally resemble prenucleolar bodies observed in higher eukaryotic cells and may represent a constituent of the normal nucleolus. Furthermore, cells under certain conditions that inhibit rRNA synthesis did not cause conversion of the nucleolus to MNBs. Thus, the role of Pol I in the maintenance of the intact nucleolar structure might include a role as a structural element in addition to (or instead of) a functional role to produce rRNA transcripts. Our study also shows that the intact nucleolar structure is not absolutely required for rRNA processing, ribosome assembly, or cell growth and that MNBs are possibly functional in rRNA processing in the Pol I deletion mutants.

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 Feedback regulation of vitamin D metabolism by 1,25-dihydroxycholecalciferol

1977 ◽  
Vol 164 (1) ◽  
pp. 83-89 ◽  
Author(s):  
K W Colston ◽  
I M A Evans ◽  
T C Spelsberg ◽  
I MacIntyre
Keyword(s):  
Vitamin D ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Subcutaneous Administration ◽  
Feedback Regulation ◽  
Rna Polymerase I ◽  
Hydroxylase Activity ◽  
Vitamin D Metabolism ◽  
Polymerase I

Many factors influence the production of 1,25(OH)2D3 (1,25-dihydroxycholecalciferol) by the kidney. One important factor seems to be feedback regulation by 1,25(OH)2D3 itself. Administration of 1,25(OH)2D3 to vitamin D-deficient chicks abolishes renal 25(OH)D3(25-hydroxycholecalciferol)1-hydroxylase activity and induces the appearance of 25(OH)D3 24-hydroxylase activity. It is likely that these effects are mediated via a nuclear effect, as they are prevented by pretreatment with actinomycin D and alpha-amanitin. Further, 1,25(OH)2D3 has a marked effect on gene transcription in the kidney cell, as assessed by measurement of RNA polymerase activities. RNA polymerase I and II activities are 80-90% inhibited by 12.5nmol of 1,25(OH)2D3 within 30min of subcutaneous administration, indicating an immediate and massive decrease in total gene transcription. By 4h RNA polymerase II activity has returned to control values, but RNA polymerase I activity is markedly enhanced. These results are consistent with the view that regulation of cholecalciferol metabolism in the kidney is associated with an effect of the active metabolite on the kidney nucleus.

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