Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non‐coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 Å resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that “braces” the helicase core. Comparative structural analyses indicate that the “brace” is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the “prong”) is an essential element for 5′‐3′ unwinding and for Sen1‐mediated transcription termination in vitro . Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro . The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5′‐3′ helicases. Synopsis Structural characterization of Ufp1‐like RNA helicase Sen1 illustrates the basis for RNA unwinding and transcription termination, and shows how disease‐linked point mutations from human ortholog senataxin impair these activities. The 1.8 Å crystal structure of the Sen1 helicase domain reveals a unique and evolutionarily conserved structural feature dubbed “brace”. The “brace” mediates extensive intramolecular interactions that are critical to maintain the fold and function of the helicase. The conserved subdomain 1C forms a “prong” that is essential for duplex unwinding and for transcription termination by Sen1 both in vitro and in vivo . Biochemical analyses of Sen1 variants harbouring senataxin mutations associated with cerebellar ataxia provide insights into the molecular basis of this disease. Graphical Abstract The core domain of RNA helicase Sen1 displays unique structural features that contribute to RNA unwinding activity and transcription termination, and explains the impact of patient mutations from the human ortholog senataxin.