scholarly journals Mechanisms of backtrack recovery by RNA polymerases I and II

2016 ◽  
Vol 113 (11) ◽  
pp. 2946-2951 ◽  
Author(s):  
Ana Lisica ◽  
Christoph Engel ◽  
Marcus Jahnel ◽  
Édgar Roldán ◽  
Eric A. Galburt ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Stochastic Theory ◽  
Rna Polymerases ◽  
Rna Cleavage ◽  
Cellular Functions ◽  
Pol Ii ◽  
Pol I ◽  
Transcript Cleavage ◽  
Polymerase I

During DNA transcription, RNA polymerases often adopt inactive backtracked states. Recovery from backtracks can occur by 1D diffusion or cleavage of backtracked RNA, but how polymerases make this choice is unknown. Here, we use single-molecule optical tweezers experiments and stochastic theory to show that the choice of a backtrack recovery mechanism is determined by a kinetic competition between 1D diffusion and RNA cleavage. Notably, RNA polymerase I (Pol I) and Pol II recover from shallow backtracks by 1D diffusion, use RNA cleavage to recover from intermediary depths, and are unable to recover from extensive backtracks. Furthermore, Pol I and Pol II use distinct mechanisms to avoid nonrecoverable backtracking. Pol I is protected by its subunit A12.2, which decreases the rate of 1D diffusion and enables transcript cleavage up to 20 nt. In contrast, Pol II is fully protected through association with the cleavage stimulatory factor TFIIS, which enables rapid recovery from any depth by RNA cleavage. Taken together, we identify distinct backtrack recovery strategies of Pol I and Pol II, shedding light on the evolution of cellular functions of these key enzymes.

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 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 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.

scholarly journals A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine.

1997 ◽  
Vol 17 (4) ◽  
pp. 1787-1795 ◽  
Author(s):  
O Gadal ◽  
S Mariotte-Labarre ◽  
S Chedin ◽  
E Quemeneur ◽  
C Carles ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Topoisomerase I ◽  
Synthetic Lethality ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Dna Topoisomerase I ◽  
Dna Topoisomerase ◽  
Pol Ii ◽  
Pol I ◽  
Polymerase I

A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.

Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II

2018 ◽  
Vol 47 (1) ◽  
pp. 425-446 ◽  
Author(s):  
Christoph Engel ◽  
Simon Neyer ◽  
Patrick Cramer
Keyword(s):  
Transcription Initiation ◽  
Messenger Rna ◽  
Rna Polymerases ◽  
Chain Elongation ◽  
Structural Studies ◽  
Pol Ii ◽  
Initiation Factors ◽  
Initiation System ◽  
Pol I ◽  
Promoter Dna

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

scholarly journals A TATA-Binding Protein Mutant Defective for TFIID Complex Formation In Vivo

1999 ◽  
Vol 19 (6) ◽  
pp. 3951-3957 ◽  
Author(s):  
Ryan T. Ranallo ◽  
Kevin Struhl ◽  
Laurie A. Stargell
Keyword(s):  
Binding Protein ◽  
Tata Binding Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Pol Ii ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I ◽  
Tfiid Complex

ABSTRACT Using an intragenic complementation screen, we have identified a temperature-sensitive TATA-binding protein (TBP) mutant (K151L,K156Y) that is defective for interaction with certain yeast TBP-associated factors (TAFs) at the restrictive temperature. The K151L,K156Y mutant appears to be functional for RNA polymerase I (Pol I) and Pol III transcription, and it is capable of supporting Gal4-activated and Gcn4-activated transcription by Pol II. However, transcription from certain TATA-containing and TATA-less Pol II promoters is reduced at the restrictive temperature. Immunoprecipitation analysis of extracts prepared after culturing cells at the restrictive temperature for 1 h indicates that the K151L,K156Y derivative is severely compromised in its ability to interact with TAF130, TAF90, TAF68/61, and TAF25 while remaining functional for interaction with TAF60 and TAF30. Thus, a TBP mutant that is compromised in its ability to form TFIID can support the response to Gcn4 but is defective for transcription from specific promoters in vivo.

The phylogenetic relations of DNA-dependent RNA polymerases of archaebacteria, eukaryotes, and eubacteria

1989 ◽  
Vol 35 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Wolfram Zillig ◽  
Hans-Peter Klenk ◽  
Peter Palm ◽  
Gabriela Pühler ◽  
Felix Gropp ◽  
...  
Keyword(s):  
Nuclear Genome ◽  
Distance Matrix ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Rna Polymerases ◽  
Matrix Analysis ◽  
Pol Ii ◽  
Phylogenetic Relations ◽  
Pol I ◽  
Pol Iii

Unrooted phylogenetic dendrograms were calculated by two independent methods, parsimony and distance matrix analysis, from an alignment of the derived amino acid sequences of the A and C subunits of the DNA-dependent RNA polymerases of the archaebacteria Sulfolobus acidocaldarius and Halobacterium halobium with 12 corresponding sequences including a further set of archaebacterial A + C subunits, eukaryotic nuclear RNA polymerases, pol I, pol II, and pol III, eubacterial β′ and chloroplast β′ and β″ subunits. They show the archaebacteria as a coherent group in close neighborhood of and sharing a bifurcation with eukaryotic pol II and (or) pol IIIA components. The most probable trees show pol IA branching off from the tree separately at a bifurcation with the eubacterial β′ lineage. The implications of these results, especially for understanding the possibly chimeric origin of the eukaryotic nuclear genome, are discussed.Key words: transcription, evolution, taxonomy, subunits, gene organization.

scholarly journals RNA Polymerase I Transcription Silences Noncoding RNAs at the Ribosomal DNA Locus in Saccharomyces cerevisiae

2009 ◽  
Vol 9 (2) ◽  
pp. 325-335 ◽  
Author(s):  
Elisa Cesarini ◽  
Francesca Romana Mariotti ◽  
Francesco Cioci ◽  
Giorgio Camilloni
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Rna Polymerase Iii ◽  
Noncoding Rnas ◽  
Rdna Locus ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Pol I ◽  
Polymerase I

ABSTRACT In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify “ribosomal” Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

scholarly journals Structural mechanism of ATP-independent transcription initiation by RNA polymerase I

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yan Han ◽  
Chunli Yan ◽  
Thi Hoang Duong Nguyen ◽  
Ashleigh J Jackobel ◽  
Ivaylo Ivanov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Polymerase I ◽  
Independent Manner ◽  
Pol Ii ◽  
Structural Mechanism ◽  
The Core ◽  
Pol I ◽  
Upstream Promoter ◽  
Polymerase I

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8 Å resolution using single-particle cryo-electron microscopy. The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from −27 to −16. Core Factor’s intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex at upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.

scholarly journals Defining the divergent enzymatic properties of RNA polymerases I and II

2020 ◽  
pp. jbc.RA120.015904
Author(s):  
Ruth Q. Jacobs ◽  
Zachariah M Ingram ◽  
Aaron L. Lucius ◽  
David A. Schneider
Keyword(s):  
Nuclear Dna ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Enzymatic Properties ◽  
Experimental Conditions ◽  
Pol Ii ◽  
Pol I ◽  
Nucleotide Addition ◽  
Addition Rate

Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult since studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single and multi-nucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes (ECs) are less stable than Pol II ECs, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.

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