Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1

The 5′→ 3′ exoribonucleases (XRNs) have important functions in transcription, RNA metabolism, and RNA interference. The recent structure of Rat1 (Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain, defining the active site of the enzyme. Xrn1 has a 510-residue segment following the conserved regions that is required for activity but is absent in Rat1. We report here the crystal structures at 2.9 Å resolution of Kluyveromyces lactis Xrn1 (residues 1–1245, E178Q mutant), alone and in complex with a Mn(2+) ion in the active site. The 510-residue segment contains four domains (D1–D4), located far from the active site. Our mutagenesis and biochemical studies demonstrate that their functional importance is due to their stabilization of the conformation of the N-terminal segment of Xrn1. These domains may also constitute a platform for interacting with protein partners of Xrn1.