Structural Basis For Interaction Of Cotranslational Chaperone RAC With The Eukaryotic 80S Ribosome

Ribosome is an intricate ribonucleoprotein complex responsible for protein biosynthesis in all living cells. After nascent polypeptides emerge from the ribosome, growing chains are in an aggregation-prone and partially folded state. Productive and preliminary folding requires ribosome-associated cotranslational chaperones to prevent non-native interactions. There are two cotranslational chaperone systems in eukaryotic cells: RAC/SSB system and NAC system. In RAC/SSB system, Zuotin(Hsp40) and Ssz(Hsp70) form a stable heterodimer termed Ribosome-Associated Complex(RAC), which bound to the ribosome through Zuotin. Zuotin serves as J-domain cochaperone of Ssb(Hsp70), specifically stimulating the ATPase activity of Ssb. Ssb binds nascent polypeptides closely to act as cotranslational chaperone only in ADP state. Importantly, emerging evidence have also suggested cotranslational chaperones are involved in translation modulation. Detailed mechanism of the work of RAC/SSB system on the ribosome is poorly understood.In this study we purified the over expressed yeast RAC and Zuotin from E.coil. Yeast 80 S ribosomes were separated by sucrose gradient centrifugation. Large datasets were collected on Titan Krios and F20 Cryo-electron microscopy. After rounds of 3D classification and refinemen t, we got high resolution structures of RAC?80S complex.RAC binds both subunits of 80 S ribosome. The contact sites on 60 S include L22, L31, H59 and H101. On the 40 S side, the contact side only involves ES12. In solutions ribosome fluctuates spontaneously between nonrotated and rotated states, and RAC could bind 80 S ribosome unrelated to the rotation state. The Middle Domain of Zuotin is composed of many charged amino acids, which form a single α-helix to mediate the electrostatic interaction between RAC an d ribosome. MD serves as a two-way signal transmitter between RAC and the ribosome: On the one hand, MD transmits the conformation change in Zuotin CTD caused by 40 S rotation to Zuotin NTD, leading J-domain getting closer to peptide tunnel exit to realize its chaperone function; On the other hand, Zuotin or RAC could limit the rotation of 40 S by MD to slow down the translation rate. Slower translation rate could benefit peptide folding. The electrical property, rigidity and optimal length of Zuotin MD are essential for yeast growth. Our structural study not only shows the binding sites of RAC on the ribosom e, but also reveals the mechanism of the translation modulation role of RAC.