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

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.

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Analysis of the Caenorhabditis elegans rpc-1 gene

10.32469/10355/4129 ◽

2005 ◽

Author(s):

◽

Qun Zheng

Keyword(s):

Gene Expression ◽

Promoter Activity ◽

Rna Polymerase Iii ◽

Transgenic Animals ◽

Rna Polymerases ◽

Pol Ii ◽

C Elegans ◽

Pol I ◽

Genes Encoding ◽

Pol Iii

In eukaryotes, two large subunits form the core catalytic structure of RNA polymerase III (Pol III), which is conserved in other RNA polymerases, Pol I and Pol II. It has been found that Pol III activity is tightly associated to cell growth. TFIII B has been shown to be one of main mediators in this process. No regulation of the Pol III largest subunit gene has been found. In C. elegans, the rpc-1 gene encodes the largest subunit of Pol III. Here, I identified two critical structural components of RPC-1, Gly644 and Gly1055, whose mutations result in larval lethal arrestment. These two amino acid residues are universally conserved in RNA polymerases, indicating their overall involvement in gene transcription mechanism. Also, I found that maternally inherited, not embryonically expressed, rpc-1 gene products survive early development. Starvation was found to suppress rpc-1 gene expression and re-feeding treatment enhances rpc-1 gene expression rapidly. No similar regulation was detected in genes encoding largest subunits of Pol I and Pol II. This is the first time that rpc-1 gene regulation has been reported. Insulin signaling may not be involved in this regulation. Also, I found that rpc-1 promoter is not ubiquitously active in C. elegans. Using the rpc-1p::gfp transgene, the rpc-1 promoter activity is only detected in a subset of neurons in the head and the tail and the intestine. While starvation silences the rpc-1 promoter activity in most tissues and cells, ASK neurons still show GFP staining in the rpc-1p::gfp transgenic animals, indicating that rpc-1 transcription in ASK neurons is continuously active under starvation conditions. Further studies suggest that TGF-[beta] signaling is involved in mediating the rpc-1 promoter activity in ASK neurons.

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Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III

10.1101/2020.01.28.923995 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yongpeng Jiang ◽

Jie Huang ◽

Kehuan Lun ◽

Boyuan Li ◽

Haonan Zheng ◽

...

Keyword(s):

Mammalian Cells ◽

Chromatin Accessibility ◽

Rna Polymerases ◽

Chromatin Interaction ◽

Transcription Inhibition ◽

Pol Ii ◽

3D Genome ◽

Chromatin Interactions ◽

Pol I ◽

Pol Iii

AbstractBackgroundThe relationship between transcription and the 3D genome organization is one of the most important questions in molecular biology, but the roles of transcription in 3D chromatin remain controversial. Multiple groups showed that transcription affects global Cohesin binding and genome 3D structures. At the same time, several studies have indicated that transcription inhibition does not affect global chromatin interactions.ResultsHere, we provide the most comprehensive evidence to date to demonstrate that transcription plays a marginal role in organizing the 3D genome in mammalian cells: 1) degraded Pol I, Pol II and Pol III proteins in mESCs, and showed their loss results in little or no changes of global 3D chromatin structures for the first time; 2) selected RNA polymerases high abundance binding sites-associated interactions and found they still persist after the degradation; 3) generated higher resolution chromatin interaction maps and revealed that transcription inhibition mildly alters small loop domains; 4) identified Pol II bound but CTCF and Cohesin unbound loops and disclosed that they are largely resistant to transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting RNA polymerases are capable of affecting the 3D genome indirectly. So, the direct and indirect effects of transcription inhibition explain the previous confusing effects on the 3D genome.ConclusionsWe conclude that Pol I, Pol II, and Pol III loss only mildly alter chromatin interactions in mammalian cells, suggesting the 3D chromatin structures are pre-established and relatively stable.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011827 ◽

2020 ◽

Vol 295 (15) ◽

pp. 4782-4795 ◽

Cited By ~ 1

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.

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Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033058 ◽

2018 ◽

Vol 47 (1) ◽

pp. 425-446 ◽

Cited By ~ 26

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.6.3951 ◽

1999 ◽

Vol 19 (6) ◽

pp. 3951-3957 ◽

Cited By ~ 8

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.

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ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

10.1101/696559 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bo Yan ◽

George Tzertzinis ◽

Ira Schildkraut ◽

Laurence Ettwiller

Keyword(s):

Rna Polymerases ◽

Transcription Start ◽

Eukaryotic Transcription ◽

Pol Ii ◽

Transcription Start Sites ◽

Cell Functions ◽

A Genome ◽

Wide Scale ◽

Nucleotide Resolution ◽

Pol Iii

AbstractMethodologies for determining eukaryotic Transcription Start Sites (TSS) rely on the selection of the 5’ canonical cap structure of Pol-II transcripts and are consequently ignoring entire classes of TSS derived from other RNA polymerases which play critical roles in various cell functions. To overcome this limitation, we developed ReCappable-seq and identified TSS from Pol-ll and non-Pol-II transcripts at nucleotide resolution. Applied to the human transcriptome, ReCappable-seq identifies Pol-II TSS with higher specificity than CAGE and reveals a rich landscape of TSS associated notably with Pol-III transcripts which have been previously not possible to study on a genome-wide scale. Novel TSS consistent with non-Pol-II transcripts can be found in the nuclear and mitochondrial genomes. By identifying TSS derived from all RNA-polymerases, ReCappable-seq reveals distinct epigenetic marks among Pol-lI and non-Pol-II TSS and provides a unique opportunity to concurrently interrogate the regulatory landscape of coding and non-coding RNA.

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Defining the divergent enzymatic properties of RNA polymerases I and II

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015904 ◽

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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Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

Journal of Bacteriology ◽

10.1128/jb.181.19.5984-5992.1999 ◽

1999 ◽

Vol 181 (19) ◽

pp. 5984-5992 ◽

Cited By ~ 40

Author(s):

Isaac K. O. Cann ◽

Sonoko Ishino ◽

Norimichi Nomura ◽

Yoshihiko Sako ◽

Yoshizumi Ishino

Keyword(s):

Amino Acid ◽

Dna Polymerase ◽

Dna Polymerases ◽

Amino Acid Sequences ◽

Pol Ii ◽

Aeropyrum Pernix ◽

Heat Stable ◽

Pol I ◽

Group I ◽

Fraction I

ABSTRACT DNA polymerase activities in fractionated cell extract ofAeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3′→5′ exonuclease and polymerase activities. The product ofpolA gene (Pol I) was aphidicolin resistant and heat stable up to 80°C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95°C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II ofPyrodictium occultum. The deduced amino acid sequence ofA. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomainCrenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

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Mechanisms of backtrack recovery by RNA polymerases I and II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517011113 ◽

2016 ◽

Vol 113 (11) ◽

pp. 2946-2951 ◽

Cited By ~ 58

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.

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