Biochemical characterization of the helicase Sen1 provides new insights into the mechanisms of non-coding transcription termination

Abstract

Pervasive transcription is widespread and needs to be controlled in order to avoid interference with gene expression. In Saccharomyces cerevisiae, the highly conserved helicase Sen1 plays a key role in restricting pervasive transcription by eliciting early termination of non-coding transcription. However, many aspects of the mechanism of termination remain unclear. In this study we characterize the biochemical activities of Sen1 and their role in termination. First, we demonstrate that the helicase domain (HD) is sufficient to dissociate the elongation complex (EC) in vitro. Both full-length Sen1 and its HD can translocate along single-stranded RNA and DNA in the 5΄ to 3΄ direction. Surprisingly, however, we show that Sen1 is a relatively poorly processive enzyme, implying that it must be recruited in close proximity to the RNA polymerase II (RNAPII) for efficient termination. We present evidence that Sen1 can promote forward translocation of stalled polymerases by acting on the nascent transcript. In addition, we find that dissociation of the EC by Sen1 is favoured by the reannealing of the DNA upstream of RNAPII. Taken together, our results provide new clues to understand the mechanism of Sen1-dependent transcription termination and a rationale for the kinetic competition between elongation and termination.

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription

“…It was recently proposed that the Mfd translocase in Escherichia coli could both nudge forward weakly paused RNAP and induce the dissociation of stalled RNAP using a release and catch-up mechanism (Le et al, 2018). However, the molecular mechanisms involved in such a release and catch-up mechanism might differ between Sen1 and Mfd, because Mfd was shown to translocate autonomously on double-stranded DNA, whereas both budding yeast and fission yeast Sen1 were shown to translocate on both single-stranded DNA and RNA, albeit at greater rate on DNA (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017). Importantly, the release and catch-up mechanism was also proposed to underlie the role of Mfd in both transcriptioncoupled repair and transcription-replication conflict resolution (Le et al, 2018) and budding yeast Sen1 has also been implicated in both transcription-coupled repair and transcriptionreplication conflict resolution (Mischo et al, 2011;Alzu et al, 2012;Brambati et al, 2018), strengthening the analogy with Mfd.…”

“…However, the mechanisms involved probably differ in both species as budding yeast Sen1 contributes to RNAP2 transcription termination as part of the Nrd1-Nab3-Sen1 (NNS) complex, which is not conserved in human cells. Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017). Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017).…”

“…In addition, both budding yeast Sen1 and human Senataxin have been implicated in the repair of DNA damage Andrews et al, 2018;Cohen et al, 2018) and in the resolution of transcription-replication conflicts (Alzu et al, 2012;Richard et al, 2013;Yü ce & West, 2013). This proposal however has been somewhat challenged by the observation that budding yeast Sen1 could directly dissociate pre-assembled RNAP2 transcription elongation complexes in vitro by translocating on the nascent RNA, even in the absence of RNA-DNA hybrids (Porrua & Libri, 2013;Han et al, 2017), suggesting that Sen1 has potentially R-loop-independent functions in the control of transcription. Current models propose that the stabilization of R-loops upon Senataxin inactivation underlies the associated transcription termination and DNA repair defects.…”

“…Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017). In addition, it has been suggested that the very low processivity of Sen1 0 s helicase activity might actually prevent it from unwinding long R-loops in vivo (Han et al, 2017). This proposal however has been somewhat challenged by the observation that budding yeast Sen1 could directly dissociate pre-assembled RNAP2 transcription elongation complexes in vitro by translocating on the nascent RNA, even in the absence of RNA-DNA hybrids (Porrua & Libri, 2013;Han et al, 2017), suggesting that Sen1 has potentially R-loop-independent functions in the control of transcription.…”

An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

“…A relatively small number of proteins are directly capable of inducing Pol II termination and include Xrn2, Pcf11, Sen1 (SETX in humans) and TTF2. Pcf11 causes dissolution of stalled elongation complexes in vitro and Sen1 is an RNA:DNA helicase that can also terminate polymerase in purified systems [28,29]. Both of these factors likely require stalled or very slow polymerases to successfully act.…”