Structural and biochemical studies of the S. cerevisiae DNA/RNA helicase Sen1

The RNA polymerase II (Pol II) is known to play a central role in transcribing all protein coding genes and non-coding RNAs (ncRNAs) in eukaryotic cells. Intriguingly, the majority of short ncRNAs are immediately degraded in the nucleus and therefore referred to as cryptic unstable transcripts (CUTs). Studies in S. cerevisiae have revealed that the Nab3-Nrd1-Sen1 (NNS) complex couples the short ncRNA transcription termination and RNA degradation by the nuclear exosome. Sen1 (252 kDa) is a well-conserved 5'→3' RNA helicase and a key player in transcription termination. In order to understand better the mechanism of termination, the helicase core domain of Sen1 (94 kDa) was expressed, purified and crystallized, and the crystal structure was solved. As shown in this work, Sen1 helicase domain has a very similar overall structure to that of Upf1-like helicases. Surprisingly, the structure reveals a unique feature, the “brace”, which fastens the accessory subdomains to RecA1 and frames the helicase in a favorable conformation for RNA binding. Moreover, structure based biochemical studies reveal that the “prong” is an essential element for 5'→3' unwinding and releasing the transcription complex from the template. Finally, I discuss the mechanism of RNA helicase translocation in the 5'→3' direction and propose a structure based model for Pol II elongation complex dissociation

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 similar to 90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 angstrom 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