Cryo-EM Study Of The Prokaryotic Small Ribosomal Subunit Maturation

Ribosome is an elegant macromolecular machine where proteins biosynthesis takesplace. In bacteria, the ribosome contains three ribosomal RNAs and over50proteins.Although the individual subunits of the ribosome can be reconstituted in vitro withmature rRNAs and r-proteins, such a reaction is inefficient in comparison to theassembly rate in vivo. The assembly of the ribosomal subunits from their components ofribosomal RNA precursors and proteins is a tightly regulated, multi-stepped process,which involves nearly thirty protein factors in vivo. The cell with a deletion of anyassembly factor shows accumulation of immature ribosomes and slow growthphenotype.RimM, RbfA and RsgA are three30S subunit assembly factors, and the deletion ofany of them causes accumulation of30S subunit assembly intermediates contain17SrRNA. In this study, firstly we present the cryo-EM structures of30S ribosome subunitbound with RsgA or RimM, comparison of our structure with other relative structuresand genetic data indicates the binding site and function role of the RsgA and RimMduring30S subunit assembly, as well as the RsgA’s role as a late stage check point.Secondly we characterize these in vivo intermediates by combination of quantitativemass spectrometry. Benefiting from cryo-electron microscopy’s ability in dealing withheterogeneous sample, we resolved the stuctures of these immature30S subunits. Theresults show that the assembly of the30S subunit is severely delayed in all theseintermediates. Further analysis indicates that the assembly of3’ domain r-protein ishighly coupled with the maturation of the16S rRNA. Based on our results andpreviously published data on immature30S, we figured out the functioal interplay ofthese assembly factors. At last, for the very first time we reveal the structure of theunprocessed terminal sequences of the17S rRNA. In summary, our work shows theuniversal role of ribosome assembly factors, on one hand, in vivo30S subunit assemblyproceeds along multiple parallel pathways, and the role of assembly factors is to directthe process to more efficient branches. On the other hand, they function as ribosomequality control system by blocking the immature ribosomal subunits from translationinitiation.