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Athar Ansari - One of the best experts on this subject based on the ideXlab platform.

  • Genomewide Analysis of Clp1 Function in Transcription in Budding Yeast
    Scientific Reports, 2017
    Co-Authors: Nadra Al-husini, Ali Sharifi, Seyed Ahmad Mousavi, Hamidreza Chitsaz, Athar Ansari
    Abstract:

    In budding yeast, the 3′ end processing of mRNA and the coupled Termination of Transcription by RNAPII requires the CF IA complex. We have earlier demonstrated a role for the Clp1 subunit of this complex in Termination and promoter-associated Transcription of CHA1. To assess the generality of the observed function of Clp1 in Transcription, we tested the effect of Clp1 on Transcription on a genomewide scale using the Global Run-On-Seq (GRO-Seq) approach. GRO-Seq analysis showed the polymerase reading through the Termination signal in the downstream region of highly transcribed genes in a temperature-sensitive mutant of Clp1 at elevated temperature. No such terminator readthrough was observed in the mutant at the permissive temperature. The poly(A)-independent Termination of Transcription of snoRNAs, however, remained unaffected in the absence of Clp1 activity. These results strongly suggest a role for Clp1 in poly(A)-coupled Termination of Transcription. Furthermore, the density of antisense transcribing polymerase upstream of the promoter region exhibited an increase in the absence of Clp1 activity, thus implicating Clp1 in promoter directionality. The overall conclusion of these results is that Clp1 plays a general role in poly(A)-coupled Termination of RNAPII Transcription and in enhancing promoter directionality in budding yeast.

  • Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach.
    Journal of Visualized Experiments, 2017
    Co-Authors: Zuzer Dhoondia, Scott Medler, Nadra Al-husini, Ricci Tarockoff, Neha Agarwal, Athar Ansari
    Abstract:

    : This manuscript describes a protocol for detecting Transcription Termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the Transcription Termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a Transcriptionally active polymerase beyond the 3' end of the gene as an indicator of a Transcription Termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the Termination signal is the one that initiated Transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3' end of the gene. Secondly, it can distinguish if the Transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

  • Gene looping facilitates TFIIH kinase-mediated Termination of Transcription.
    Scientific Reports, 2015
    Co-Authors: Scott Medler, Athar Ansari
    Abstract:

    TFIIH is a general Transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of Transcription is well established. Here we report a novel role of Kin28 in the Termination of Transcription. We show that RNAPII reads through a Termination signal upon kinase inhibition. Furthermore, the recruitment of Termination factors towards the 3′ end of a gene was compromised in the kinase mutant, thus confirming the Termination defect. A concomitant decrease in crosslinking of Termination factors near the 5′ end of genes was also observed in the kinase-defective mutant. Simultaneous presence of Termination factors towards both the ends of a gene is indicative of gene looping; while the loss of Termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the Termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the Termination of Transcription in budding yeast.

  • srb5 med18 mediated Termination of Transcription is dependent on gene looping
    Journal of Biological Chemistry, 2013
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the Termination of Transcription for a subset of genes in yeast. Srb5/Med18 could affect Termination either indirectly by modulating CTD-Ser2 phosphorylation near the 3′ end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser2 phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3′ end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5− cells. Furthermore, Srb5-dependent Termination of Transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated Termination of Transcription, and the looped gene architecture may have a general role in Termination of Transcription in budding yeast.

  • Srb5/Med18-mediated Termination of Transcription Is Dependent on Gene Looping
    Journal of Biological Chemistry, 2013
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the Termination of Transcription for a subset of genes in yeast. Srb5/Med18 could affect Termination either indirectly by modulating CTD-Ser2 phosphorylation near the 3′ end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser2 phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3′ end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5− cells. Furthermore, Srb5-dependent Termination of Transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated Termination of Transcription, and the looped gene architecture may have a general role in Termination of Transcription in budding yeast.

Banupriya Mukundan - One of the best experts on this subject based on the ideXlab platform.

  • srb5 med18 mediated Termination of Transcription is dependent on gene looping
    Journal of Biological Chemistry, 2013
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the Termination of Transcription for a subset of genes in yeast. Srb5/Med18 could affect Termination either indirectly by modulating CTD-Ser2 phosphorylation near the 3′ end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser2 phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3′ end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5− cells. Furthermore, Srb5-dependent Termination of Transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated Termination of Transcription, and the looped gene architecture may have a general role in Termination of Transcription in budding yeast.

  • Srb5/Med18-mediated Termination of Transcription Is Dependent on Gene Looping
    Journal of Biological Chemistry, 2013
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    We have earlier demonstrated the involvement of Mediator subunit Srb5/Med18 in the Termination of Transcription for a subset of genes in yeast. Srb5/Med18 could affect Termination either indirectly by modulating CTD-Ser2 phosphorylation near the 3′ end of a gene or directly by physically interacting with the cleavage and polyadenylation factor or cleavage factor 1 (CF1) complex and facilitating their recruitment to the terminator region. Here, we show that the CTD-Ser2 phosphorylation pattern on Srb5/Med18-dependent genes remains unchanged in the absence of Srb5 in cells. Coimmunoprecipitation analysis revealed the physical interaction of Srb5/Med18 with the CF1 complex. No such interaction of Srb5/Med18 with the cleavage and polyadenylation factor complex, however, could be detected. The Srb5/Med18-CF1 interaction was not observed in the looping defective sua7-1 strain. Srb5/Med18 cross-linking to the 3′ end of genes was also abolished in the sua7-1 strain. Chromosome conformation capture analysis revealed that the looped architecture of Srb5/Med18-dependent genes was abrogated in srb5− cells. Furthermore, Srb5-dependent Termination of Transcription was compromised in the looping defective sua7-1 cells. The overall conclusion of these results is that gene looping plays a crucial role in Srb5/Med18 facilitated Termination of Transcription, and the looped gene architecture may have a general role in Termination of Transcription in budding yeast.

  • novel role for mediator complex subunit srb5 med18 in Termination of Transcription
    Journal of Biological Chemistry, 2011
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    Mediator complex functions at the recruitment as well as the post-recruitment steps of Transcription. Here we provide evidence for a novel role of Mediator in Termination of Transcription. Mediator subunit Srb5/Med18 cross-links to the 5′ and 3′ ends of INO1 and CHA1. In srb5− cells, recruitment of TATA-binding protein (TBP) and Transcription factor IIB (TFIIB) onto the promoter of these genes remained unaffected, but cross-linking of the cleavage-polyadenylation factors Rna15 and Pta1 toward the 3′ end of genes was compromised. In these cells, RNA polymerase II accumulated near the 3′ end of genes and beyond. Transcription run-on analysis confirmed a Transcription readthrough phenotype in the absence of Srb5/Med18. These results strongly suggest that Mediator subunit Srb5/Med18 is required for proper Termination of Transcription of a subset of genes in budding yeast.

  • Novel role for mediator complex subunit Srb5/Med18 in Termination of Transcription.
    Journal of Biological Chemistry, 2011
    Co-Authors: Banupriya Mukundan, Athar Ansari
    Abstract:

    Mediator complex functions at the recruitment as well as the post-recruitment steps of Transcription. Here we provide evidence for a novel role of Mediator in Termination of Transcription. Mediator subunit Srb5/Med18 cross-links to the 5′ and 3′ ends of INO1 and CHA1. In srb5− cells, recruitment of TATA-binding protein (TBP) and Transcription factor IIB (TFIIB) onto the promoter of these genes remained unaffected, but cross-linking of the cleavage-polyadenylation factors Rna15 and Pta1 toward the 3′ end of genes was compromised. In these cells, RNA polymerase II accumulated near the 3′ end of genes and beyond. Transcription run-on analysis confirmed a Transcription readthrough phenotype in the absence of Srb5/Med18. These results strongly suggest that Mediator subunit Srb5/Med18 is required for proper Termination of Transcription of a subset of genes in budding yeast.

Daniel Reines - One of the best experts on this subject based on the ideXlab platform.

  • A fluorescent assay for the genetic dissection of the RNA polymerase II Termination machinery.
    Methods, 2019
    Co-Authors: Daniel Reines
    Abstract:

    Abstract RNA polymerase II is a highly processive enzyme that synthesizes mRNAs and some non-protein coding RNAs. Termination of Transcription, which entails release of the transcript and disengagement of the polymerase, requires an active process. In yeast, there are at least two multi-protein complexes needed for Termination of Transcription, depending upon which class of RNAs are being acted upon. In general, the two classes are relatively short non-coding RNAs (e.g. snoRNAs) and relatively long mRNAs, although there are exceptions. Here, a procedure is described in which defective Termination can be detected in living cells, resulting in a method that allows strains with mutations in Termination factors or cis-acting sequences, to be identified and recovered. The strategy employs a reporter plasmid with a galactose inducible promoter driving Transcription of green fluorescent protein which yields highly fluorescent cells. When a test terminator is inserted between the promoter and the fluorescent protein reading frame, cells fail to fluoresce. Mutant strains that have lost Termination capability, so called terminator-override mutants, gain expression of the fluorescent protein and can be collected by fluorescence activated cell sorting. The strategy is robust since acquisition of fluorescence is a positive trait that has a low probability of happening adventitiously. Live mutant cells can easily be cloned from the population of positive candidates. Flow sorting is a sensitive, high-throughput detection step capable of discovering spontaneous mutations in yeast with high fidelity.

  • Termination of Transcription of short noncoding rnas by rna polymerase ii
    Annual Review of Biochemistry, 2015
    Co-Authors: Karen M Arndt, Daniel Reines
    Abstract:

    The RNA polymerase II Transcription cycle is often divided into three major stages: initiation, elongation, and Termination. Research over the last decade has blurred these divisions and emphasized the tightly regulated transitions that occur as RNA polymerase II synthesizes a transcript from start to finish. Transcription Termination, the process that marks the end of Transcription elongation, is regulated by proteins that interact with the polymerase, nascent transcript, and/or chromatin template. The failure to terminate Transcription can cause accumulation of aberrant transcripts and interfere with Transcription at downstream genes. Here, we review the mechanism, regulation, and physiological impact of a Termination pathway that targets small noncoding transcripts produced by RNA polymerase II. We emphasize the Nrd1–Nab3–Sen1 pathway in yeast, in which the process has been extensively studied. The importance of understanding small RNA Termination pathways is underscored by the need to control noncoding Transcription in eukaryotic genomes.

Marc Boudvillain - One of the best experts on this subject based on the ideXlab platform.

  • a fluorogenic assay to monitor rho dependent Termination of Transcription
    Biochemistry, 2019
    Co-Authors: Cedric Nadiras, Annie Schwartz, Marc Boudvillain, Mildred Delaleau, Emmanuel Margeat
    Abstract:

    Transcription Termination mediated by the ring-shaped, ATP-dependent Rho motor is a multipurpose regulatory mechanism specific to bacteria and constitutes an interesting target for the development of new antibiotics. Although Rho-dependent Termination can punctuate gene expression or contribute to the protection of the genome at hundreds of sites within a given bacterium, its exact perimeter and site- or species-specific features remain insufficiently characterized. New advanced approaches are required to explore thoroughly the diversity of Rho-dependent terminators and the complexity of associated mechanisms. Current in vitro analyses of Rho-dependent Termination rely on radiolabeling, gel electrophoresis, and phosphorimaging of Transcription reaction products and are thus hazardous, inconvenient, and low-throughput. To address these limitations, we have developed the first in vitro assay using a fluorescence detection modality to study Rho-dependent Transcription Termination. This powerful experimental tool accurately estimates terminator strengths in a matter of minutes and is optimized for a microplate reader format allowing multiplexed characterization of putative terminator sequences and mechanisms or high-throughput screening of new drugs targeting Rho-dependent Termination.

  • a multivariate prediction model for rho dependent Termination of Transcription
    Nucleic Acids Research, 2018
    Co-Authors: Cedric Nadiras, Eric Eveno, Annie Schwartz, Nara Figueroabossi, Marc Boudvillain
    Abstract:

    : Bacterial Transcription Termination proceeds via two main mechanisms triggered either by simple, well-conserved (intrinsic) nucleic acid motifs or by the motor protein Rho. Although bacterial genomes can harbor hundreds of Termination signals of either type, only intrinsic terminators are reliably predicted. Computational tools to detect the more complex and diversiform Rho-dependent terminators are lacking. To tackle this issue, we devised a prediction method based on Orthogonal Projections to Latent Structures Discriminant Analysis [OPLS-DA] of a large set of in vitro Termination data. Using previously uncharacterized genomic sequences for biochemical evaluation and OPLS-DA, we identified new Rho-dependent signals and quantitative sequence descriptors with significant predictive value. Most relevant descriptors specify features of transcript C>G skewness, secondary structure, and richness in regularly-spaced 5'CC/UC dinucleotides that are consistent with known principles for Rho-RNA interaction. Descriptors collectively warrant OPLS-DA predictions of Rho-dependent Termination with a ∼85% success rate. Scanning of the Escherichia coli genome with the OPLS-DA model identifies significantly more Termination-competent regions than anticipated from transcriptomics and predicts that regions intrinsically refractory to Rho are primarily located in open reading frames. Altogether, this work delineates features important for Rho activity and describes the first method able to predict Rho-dependent terminators in bacterial genomes.

  • evaluating the effect of small rnas and associated chaperones on rho dependent Termination of Transcription in vitro
    Methods of Molecular Biology, 2018
    Co-Authors: Cedric Nadiras, Annie Schwartz, Mildred Delaleau, Marc Boudvillain
    Abstract:

    : Besides their well-known postTranscriptional effects on mRNA translation and decay, sRNAs and associated RNA chaperones (e.g., Hfq, CsrA) sometimes regulate gene expression at the Transcriptional level. In this case, the sRNA-dependent machinery modulates the activity of the Transcription Termination factor Rho, a ring-shaped RNA translocase/helicase that dissociates Transcription elongation complexes at specific loci of the bacterial genome. Here, we describe biochemical assays to detect Rho-dependent Termination signals in genomic regions of interest and to assess the effects of sRNAs and/or associated RNA chaperones on such signals.

  • Transcription Termination variations on common themes
    Trends in Genetics, 2016
    Co-Authors: Odil Porrua, Marc Boudvillain, Domenico Libri
    Abstract:

    Transcription initiates pervasively in all organisms, which challenges the notion that the information to be expressed is selected mainly based on mechanisms defining where and when Transcription is started. Together with post-Transcriptional events, Termination of Transcription is essential for sorting out the functional RNAs from a plethora of Transcriptional products that seemingly have no use in the cell. But terminating Transcription is not that easy, given the high robustness of the elongation process. We review here many of the strategies that prokaryotic and eukaryotic cells have adopted to dismantle the elongation complex in a timely and efficient manner. We highlight similarities and diversity, underlying the existence of common principles in a diverse set of functionally convergent solutions.

A. Limanskii - One of the best experts on this subject based on the ideXlab platform.

  • Study of elongation complexes for T7 RNA polymerase
    Biophysics, 2012
    Co-Authors: O. Limanskaya, A. Limanskii
    Abstract:

    Complexes of bacteriophage T7 RNA polymerase with a DNA template for Transcription elongation were visualized by atomic force microscopy. Images for complexes of T7 RNA polymerase with terminal fragments of DNA template were obtained for single molecules. Complexes of a single DNA template molecule with several T7 RNA polymerase molecules corresponding to stages of initiation, elongation and Termination of Transcription were visualized under the elimination of unspecific DNA-protein binding. Immobilized on the amino mica RNA transcripts form rod-like condensed structures. Detailes of specific and unspecific complex formation for the T7 RNA polymerase-DNA system during initiation and Transcription elongation are discussed.