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Characterization of the Mechanisms of Transcription Termination by the Helicase Sen1

  • Odil PorruaEmail author
  • Domenico Libri
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

In vitro transcription systems have been widely used to study all the steps of transcription from initiation to termination and many transcription-coupled processes. Here we describe an in vitro transcription-termination assay that we have used for the analysis of the mechanism of termination by the yeast helicase Sen1. In this system, we use highly purified proteins to assemble ternary elongation complexes (RNA polymerase, DNA template, and nascent RNA) on biotinylated DNA that is subsequently immobilized on streptavidin beads. After allowing transcription by the addition of nucleotides, the termination events can be detected and quantified by comparing the amounts of polymerases and transcripts released from the DNA templates in reactions performed in the absence or in the presence of purified Sen1. By modifying different parameters of the assay, this technique allows the study of several aspects of the termination reaction.

Key words

In vitro transcription RNA and DNA helicases Sen1 Transcription termination RNAPII purification Ternary elongation complex Promoter-independent assembly of elongation complexes 

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Centre de Génétique MoléculaireCNRS UPR3404Gif sur YvetteFrance
  2. 2.Institut Jacques Monod, CNRS UMR 7592Université Paris Diderot, Sorbonne Paris CitéParisFrance

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