scholarly journals Biased Pol II fidelity contributes to conservation of functional domains in the Potato spindle tuber viroid genome

2020 ◽  
Vol 16 (12) ◽  
pp. e1009144
Author(s):  
Jian Wu ◽  
David M. Bisaro
Keyword(s):  
Rna Polymerase Ii ◽  
De Novo ◽  
Potato Spindle Tuber Viroid ◽  
Mutation Rates ◽  
Wild Type ◽  
Pol Ii ◽  
Synonymous Mutations ◽  
Novel Strategy ◽  
Critical Regions

Accurate calculation of mutation rates for viruses and viroids is necessary for evolutionary studies and to evaluate adaptation potential. However, estimation of in vivo mutation rates is complicated by selection, which leads to loss or proliferation of certain mutations. To minimize this concern, lethal mutations, including nonsense and non-synonymous mutations, have been used to determine mutation rates for several viruses and viroids, including Potato spindle tuber viroid (PSTVd). However, this approach has limitations, including focus on a relatively small number of genome sites and the possibility that mutations may not actually be lethal or may be maintained by wild type individuals. To avoid selection bias altogether, we sequenced minus-strand PSTVd dimers from concatemeric replication intermediates. The underlying rationale is that mutations found in only one of the monomers were likely generated de novo during RNA polymerase II (Pol II) transcription of the circular plus-strand RNA genome. This approach yielded an apparent Pol II error rate of ~1/1837 nucleotides per transcription cycle, and an estimated mutation rate of ~1/919 nucleotides for a single replication cycle. Remarkably, de novo mutations were nearly absent from the most conserved, replication-critical regions of the PSTVd genome, suggesting that sequence conservation is a consequence of both essential function and template optimization for greater Pol II fidelity. Such biased fidelity may constitute a novel strategy to ensure population success while allowing abundant sampling of sequence space in other genome regions. Comparison with variants in progeny populations derived from a cloned, wild type PSTVd master sequence revealed that most de novo mutations were lost through selection.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals Saccharomyces cerevisiae Transcription Elongation Mutants Are Defective in PUR5 Induction in Response to Nucleotide Depletion

2000 ◽  
Vol 20 (20) ◽  
pp. 7427-7437 ◽  
Author(s):  
Randal J. Shaw ◽  
Daniel Reines
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Normal Growth ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Luciferase Reporter Gene ◽  
Pol Ii

ABSTRACT IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective inPUR5 induction. The inability to fully inducePUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5′ untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.

scholarly journals Evidence Supporting That RNA Polymerase II Catalyzes De Novo Transcription Using Potato Spindle Tuber Viroid Circular RNA Templates

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 371
Author(s):  
Shachinthaka D. Dissanayaka Mudiyanselage ◽  
Ying Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
De Novo ◽  
Unit Length ◽  
Potato Spindle Tuber Viroid ◽  
Circular Rna ◽  
Asymmetric Rolling ◽  
Pol Ii ◽  
Rolling Circle ◽  
Dna And Rna

Transcription is a fundamental process that mediates the interplay between genetic information and phenotype. Emerging evidence indicates that RNA polymerase II (Pol II) can catalyze transcription using both DNA and RNA templates. It is well established that Pol II initiates de novo transcription on DNA templates. However, it is unclear whether Pol II performs de novo transcription or relies on primers for initiation (primed transcription) on RNA templates. Using potato spindle tuber viroid (PSTVd) as a model, we presented evidence showing that circular PSTVd templates are critical for the synthesis of longer-than-unit-length (−)-strand products, which supports the de novo transcription based on the asymmetric rolling circle model of PSTVd replication. We further showed that the crucial factor for primed transcription, transcription factor IIS (TFIIS), is dispensable for PSTVd replication in cells. Together, our data support the de novo transcription on PSTVd RNA templates catalyzed by Pol II. This result has significant implications in understanding the mechanism and machinery underlying Pol II-catalyzed transcription using other RNA templates.

Abstract 355: In Vivo Targeting Of GTF2B Selectively Inhibits Transcription Of Cardiomyopathy-related Genes And Partially Blunts Development Of Cardiac Hypertrophy

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Danish Sayed ◽  
Zhi Yang ◽  
Minzhen He ◽  
Maha Abdellatif
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Splicing ◽  
De Novo ◽  
Transcriptional Profiling ◽  
Essential Genes ◽  
Pol Ii ◽  
Expression Of Genes ◽  
Essential Components

Transcriptional profiling of cardiac genome during hypertrophy identified two categories of genes with distinct modes of regulation. The first set of genes involved in the cells essential functions (e.g. RNA splicing) and whose transcription is expected to be incremental and contribute to the increasing cardiac mass is regulated by promoter clearance of RNA polymerase II (pol II). On the other hand, the second set that include genes with specialized function and show a robust increase in expression upon growth stimulus (cytoskeletal, extracellular matrix) are regulated by de novo pol II recruitment to promoters. Our goal was to identify the transcriptional mechanisms that distinguish these two sets of genes and then to selectively inhibit those that participate in contractile dysfunction, while preserving the expression of genes necessary for essential functions. General Transcription factor IIB (GTF2B), is one of the essential components of transcription machinery and is required for pol II recruitment. Thus, we hypothesized that inhibition of GTF2B would result in inhibition of only the specialized genes, sparing the essential genes. Our in vitro results with shRNA mediated inhibition of GTF2B in hypertrophying neonatal myocytes showed decreased expression of genes that required de novo pol II recruitment for transcription (eg. ACTA1), while no change was observed in the genes regulated by promoter clearance of pol II (Vdac1). Similarly, preliminary results with in vivo knockdown of GTF2B (~80% reduction in mRNA and ~36% in protein) via intravenous injection of modified antisense oligo in mice subjected to transaortic coarctation (TAC) showed inhibition of only cardiomyopathy-related genes that require pol II recruitment (ANF), while expression of essential genes (Vdac1) remained unchanged. Inhibition of GTF2B restricted increase in TAC-induced heart wt to 9%, compared to 29% in TAC hearts with control oligo. Echocardiography showed partial normalization of ejection fraction with GTF2B inhibitor during TAC from 61.5% to 66.4% compared to sham hearts with 71%. Thus, we conclude that by targeting GTF2B we can selectively restrict the expression of detrimental genes during hypertrophy, thereby delaying the onset of cardiac dysfunction and failure

scholarly journals The FACT Complex Travels with Elongating RNA Polymerase II and Is Important for the Fidelity of Transcriptional Initiation In Vivo

2003 ◽  
Vol 23 (22) ◽  
pp. 8323-8333 ◽  
Author(s):  
Paul B. Mason ◽  
Kevin Struhl
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Tata Binding Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Transcriptional Initiation ◽  
Pol Ii ◽  
Coding Regions

ABSTRACT The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors. In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II. Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3′ portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions. Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters. Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized. We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.

scholarly journals Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries

2002 ◽  
Vol 1 (3) ◽  
pp. 448-457 ◽  
Author(s):  
Toshimitsu Takagi ◽  
Eun-Jung Cho ◽  
Rozmin T. K. Janoo ◽  
Vladimir Polodny ◽  
Yasutaka Takase ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Rna Polymerase Ii ◽  
Subunit Interactions ◽  
Pol Ii ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Enzyme Subunits ◽  
Carboxyl Terminal

ABSTRACT The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1 +) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals Independent Recruitment of Mediator and SAGA by the Activator Met4

2006 ◽  
Vol 26 (8) ◽  
pp. 3149-3163 ◽  
Author(s):  
Christophe Leroy ◽  
Laëtitia Cormier ◽  
Laurent Kuras
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Rna Polymerase Ii ◽  
Gene Network ◽  
Pol Ii ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Target Promoters ◽  
Sulfur Containing

ABSTRACT Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification.

scholarly journals CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

2007 ◽  
Vol 27 (5) ◽  
pp. 1631-1648 ◽  
Author(s):  
Igor Chernukhin ◽  
Shaharum Shamsuddin ◽  
Sung Yun Kang ◽  
Rosita Bergström ◽  
Yoo-Wook Kwon ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Ctcf Binding ◽  
Pol Ii ◽  
Genome Wide ◽  
Target Sites ◽  
On Chip

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

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