Termination of non‐coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD‐mimicking region in Sen1

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non‐coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non‐coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy‐terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD‐interacting domain of Nrd1, an RNA‐binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1‐dependent termination strictly requires CTD recognition by the N‐terminal domain of Sen1. We provide evidence that the Sen1‐CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein–protein interactions that control termination of non‐coding transcription by Sen1.