Structural basis of ribosomal 30S subunit degradation by RNase R

Protein synthesis is a major energy-consuming process of the cell, which requires controlled production and turnover of ribosomes. While the last years have seen major advances in our understanding of ribosome biogenesis, structural insight into the degradation of ribosomes has been lacking. Here we present native structures of two distinct small ribosomal 30S subunit degradation intermediates associated with the 3’ to 5’ exonuclease, RNase R. The structures reveal that RNase R binds initially to the 30S platform to facilitate degradation of the functionally important anti-Shine-Dalgarno sequence and decoding site helix 44. RNase R then encounters a roadblock when it reaches the neck region of the 30S, which is overcome by a major structural rearrangement of the 30S head, involving loss of ribosomal proteins. RNase R parallels this movement, relocating to the decoding site, by using its N-terminal helix-turn-helix domain as an anchor. In vitro degradation assays suggest that head rearrangement poses a major kinetic barrier for RNase R, but also that the enzyme alone is sufficient for complete 30S degradation. Collectively, our results provide a mechanistic basis for RNase R-mediated 30S degradation and reveal that RNase R targets orphaned 30S subunits using a dynamic anchored binding site switching mechanism. Rnase R is a 3' exonuclease which degrades the 16S rRNA within the 30S ribosomal subunit. To compare the truncation site from an in vivo pull down and an in vitro rection whereby recombinant Rnase R was mixed with isolated 30S subunits, we sequenced the extracted RNA in a high-througput reaction. For the in vivo sequencing the RNA from three independent IPs of endogenously Flag-tagged RNase R was extracted using Trizol. The in vitro sequencing was also carried out over three independent reactions.