Study Of The Large Ribosomal Subunit Assembly In Yeast

Protein biosynthesis is catalyzed by the ribosome in all living organisms.Thousands of ribosomes must be synthesized per minute to maintain and update the tremendous protein inventory in rapidly growing cells.Ribosome consists of large and small subunit which is composed by ribosomal RNA and protein.The function of SBDS is cooperate with elongation factor 1-like(Efl1)to release the eukaryotic initiation factor 6(eIF6)from the late-stage cytoplasmic pre-60 S.The human Shwachman-Diamond syndrome(SDS)is an autosomal recessive disease which is caused by mutations in the ribosomal subunit maturation factor SBDS.Our data reveal that Sdo1 interacts with the mature 60 S subunit though its domain I and II in vitro and induces the 60 S subunits dimerization with the formation of a stable 2:2 dimer.Structural analysis shows that Sdo1 bind to the ribosomal P-site and contact to H69 and H38.The flexiblity of Sdo1 on the 60 S subunit and its binding position suggests the role of Sdo1 in monitoring the conformational maturation status of the ribosomal P-site.The nuclear export of pre-ribosomal particles is a key irreversible step during ribosome biogenesis.Nmd3,a highly conserved protein in eukaryotes,is an adaptor protein required for the export of pre-60 S particles.The Cterminal of Nmd3 contains a nuclear export signal sequence(NES)which is responsible for recruiting Crm1.Here,we characterize the late-stage cytoplasmic pre-60 S particles purified from Saccharomyces cerevisiae using epitope-tagged Nmd3 with cryo-electron microscopy.The Nmd3-particles contains four assembling factors Nmd3,Lsg1,Tif6 and Reh1,and lacks the ribosomal protein uL16,u L10,uL11,eL40 and eL41.Nmd3 and Lsg1 are next to each other near the peptidyl-transferase center(PTC)and distinguish the status of PTC through uL10.Reh1 blocks the peptide tunnel exit with its C-terminus inserted into the tunnel.The structural observations highlight the final maturation steps of cytoplasmic pre-60 S particles and suggest the molecular roles of these factors.These results suggested Nmd3 acts as structural checkpoint in the PTC assembly.Nmd3 is released from pre-60 S with the cooperation of RPL10 and Lsg1 after transporting to the cytoplasm.