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Single-molecule characterization of extrinsic transcription termination by Sen1 helicase

Author

Listed:
  • S. Wang

    (PSL Research University
    Sorbonne Paris Cité)

  • Z. Han

    (Sorbonne Paris Cité)

  • D. Libri

    (Sorbonne Paris Cité)

  • O. Porrua

    (Sorbonne Paris Cité)

  • T. R. Strick

    (PSL Research University
    Sorbonne Paris Cité
    Ligue Contre le Cancer)

Abstract

Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.

Suggested Citation

  • S. Wang & Z. Han & D. Libri & O. Porrua & T. R. Strick, 2019. "Single-molecule characterization of extrinsic transcription termination by Sen1 helicase," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-09560-9
    DOI: 10.1038/s41467-019-09560-9
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    Cited by:

    1. Ying Xiong & Weijing Han & Chunhua Xu & Jing Shi & Lisha Wang & Taoli Jin & Qi Jia & Ying Lu & Shuxin Hu & Shuo-Xing Dou & Wei Lin & Terence R. Strick & Shuang Wang & Ming Li, 2024. "Single-molecule reconstruction of eukaryotic factor-dependent transcription termination," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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