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A bacterial-like mechanism for transcription termination by the Sen1p helicase in budding yeast

Nature Structural & Molecular Biology volume 20, pages 884–891 (2013)Cite this article

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Abstract

Transcription termination is essential to generate functional RNAs and to prevent disruptive polymerase collisions resulting from concurrent transcription. The yeast Sen1p helicase is involved in termination of most noncoding RNAs transcribed by RNA polymerase II (RNAPII). However, the mechanism of termination and the role of this protein have remained enigmatic. Here we address the mechanism of Sen1p-dependent termination by using a highly purified in vitro system. We show that Sen1p is the key enzyme of the termination reaction and reveal features of the termination mechanism. Like the bacterial termination factor Rho, Sen1p recognizes the nascent RNA and hydrolyzes ATP to dissociate the elongation complex. Sen1p-dependent termination is highly specific and, notably, does not require the C-terminal domain of RNAPII. We also show that termination is inhibited by RNA-DNA hybrids. Our results elucidate the role of Sen1p in controlling pervasive transcription.

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Figure 1: Purified Sen1p elicits transcription termination in vitro.
Figure 2: Sen1-mediated transcription termination is primed by the nascent RNA.
Figure 3: Sen1-mediated termination requires ATP hydrolysis.
Figure 4: The Sen1p-related RNA helicase Upf1p cannot terminate RNAPII transcription.
Figure 5: Analysis of the roles of R loops and helicase activity in Sen1-dependent termination.
Figure 6: Sen1-dependent termination is restricted to eukaryotic RNAPII but does not require the CTD.
Figure 7: Model for NNS-dependent termination in vivo.

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Acknowledgements

We wish to thank E. Lehman and P. Cramer (Gene Center, Munich, Germany) for initial help with the in vitro transcription assays and for the gift of the His-tagged Rpb3 strain35 and plasmid pET21b(+)-Rpb4-7; F. Fiorini and H. Le Hir (Ecole Normale Supérieure, Paris, France) for the gift of Upf1 variants and help with the ATPase assays; M. Swanson (University of Florida, Gainesville, Florida, USA) for the gift of anti-Nab3p antibody; T. Villa for constructing the DL1774 strain; K. Tanner, M. Boudavillain, T. Villa, E. Pearson, C. Moore and T. H. Jensen for critical reading of the manuscript; M.-C. Daugeron, T. Villa and the other members of the Libri lab for helpful discussions; and S. Buratowski, E. Pearson and C. Moore for sharing results before publication. This work was supported by the Danish National Research Foundation, the French Agence Nationale pour la Recherche (ANR-08-Blan-0038-01, to D.L.) and the French Centre National de la Recherche Scientifique. O.P. was supported by a European Molecular Biology Organization Long Term fellowship and is presently funded by the French Fondation pour la Recherche Médicale. This research was carried out within the scope of the Associated European Laboratory 'Laboratory of Nuclear RNA Metabolism'.

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  1. Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, Gif sur Yvette, France

    Odil Porrua & Domenico Libri

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O.P. designed and performed experiments and wrote the paper. D.L. designed experiments and wrote the paper.

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Correspondence to Odil Porrua or Domenico Libri.

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Porrua, O., Libri, D. A bacterial-like mechanism for transcription termination by the Sen1p helicase in budding yeast. Nat Struct Mol Biol 20, 884–891 (2013). https://doi.org/10.1038/nsmb.2592

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