Structural and Functional Analysis of the Archaeal Endonuclease Nob1

Eukaryotic ribosome biogenesis depends on the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, was identified as being responsible for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the nascent subunit into the cytoplasm. The same holds true for Nob1 from the Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the pre-ribosomal RNA in a manganese dependent manner. The structure of PhNob1 solved by NMR-spectroscopy revealed a PIN-domain common in many nucleases and a zinc ribbon domain which are structurally connected by a flexible linker. We show that the PIN-domain binds to substrate RNAs with rather low affinity while the zinc ribbon domain is sufficient to bind helix 40 of the small subunit rRNA with high affinity. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site. This mode of action is reminiscent of many RNA helicases which also contain a high affinity RNA-binding domain for anchoring and helicase module with lower affinity and specificity.