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A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting

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      Abstract

      Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.

      Abstract

      HIV-1 viral gene expression stochastically switches between active and inactive states. Here, using improved single molecule RNA microscopy, the authors show that HIV-1 RNA stochastic transcription is achieved by groups of closely spaced polymerases, and is regulated by Mediator and TBP at different time scales.

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      Most cited references 61

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      Mfold web server for nucleic acid folding and hybridization prediction.

       M Zuker (2003)
      The abbreviated name, 'mfold web server', describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces), the server circumvents the problem of portability of this software. Detailed output, in the form of structure plots with or without reliability information, single strand frequency plots and 'energy dot plots', are available for the folding of single sequences. A variety of 'bulk' servers give less information, but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/mfold. This URL will be referred to as 'MFOLDROOT'.
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        Nature, nurture, or chance: stochastic gene expression and its consequences.

        Gene expression is a fundamentally stochastic process, with randomness in transcription and translation leading to cell-to-cell variations in mRNA and protein levels. This variation appears in organisms ranging from microbes to metazoans, and its characteristics depend both on the biophysical parameters governing gene expression and on gene network structure. Stochastic gene expression has important consequences for cellular function, being beneficial in some contexts and harmful in others. These situations include the stress response, metabolism, development, the cell cycle, circadian rhythms, and aging.
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          Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.

          RNA polymerases are highly regulated molecular machines. We present a method (global run-on sequencing, GRO-seq) that maps the position, amount, and orientation of transcriptionally engaged RNA polymerases genome-wide. In this method, nuclear run-on RNA molecules are subjected to large-scale parallel sequencing and mapped to the genome. We show that peaks of promoter-proximal polymerase reside on approximately 30% of human genes, transcription extends beyond pre-messenger RNA 3' cleavage, and antisense transcription is prevalent. Additionally, most promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene. This divergent polymerase is associated with active genes but does not elongate effectively beyond the promoter. These results imply that the interplay between polymerases and regulators over broad promoter regions dictates the orientation and efficiency of productive transcription.
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            Author and article information

            Affiliations
            [1 ]Institut de Génétique Moléculaire de Montpellier, CNRS UMR5535, 1919, route de Mende , 34293 Montpellier Cedex 5, France
            [2 ]Unité Imagerie et Modélisation, Département Biologie Cellulaire et Infections, Institut Pasteur and CNRS UMR 3691, 28, rue du Docteur Roux , 75015 Paris, France
            [3 ]Laboratoire de Physique Théorique de la Matière Condensée; CNRS UMR 7600, UPMC-Paris 6, Sorbonne Universités, 4 place Jussieu , 75252 Paris Cedex 5, France
            [4 ]BioCampus Montpellier, CNRS UMS3426, 141, rue de la Cardonille , 34094 Montpellier Cedex 5, France
            [5 ]Ecole Normale Supérieure, CNRS UMR 8197 Paris, France
            Author notes
            [*]

            Present address: Department of Molecular and Cell Biology, University of California in Berkeley, Berkeley, California 94720, USA.

            Journal
            Nat Commun
            Nat Commun
            Nature Communications
            Nature Publishing Group
            2041-1723
            27 July 2016
            2016
            : 7
            27461529
            4974459
            ncomms12248
            10.1038/ncomms12248
            Copyright © 2016, The Author(s)

            This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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