Unsupervised Voxel-based Segmentation reveals a Landscape of Bacterial Ribosome Large Subunit Early Assembly

Ribosome biogenesis is a complex but efficient process in rapidly growing bacteria, where assemble a functional 70S ribosome takes ~ 2 min, involving participation of 3 rRNAs, 50 r-proteins and dozens of assembly factors. In vitro reconstitution using different subsets of large subunit (50S, LSU) proteins with rRNAs, pioneered by Nierhaus lab, resulted in the Nierhaus assembly map, embodying the cooperativity and dependency for binding of LSU r-proteins to 23S rRNA. Critically absent from the Nierhaus map is the underlying folding of the rRNA that creates the binding sites for the r-proteins. In addition, the relationship of the observed cooperativity in vitro to the co-transcriptional assembly in cells remains to be determined. Pre-50S intermediates accumulate at low temperature in ΔdeaD, a DEAD-box helicase implicated in 50S assembly. We solved 21 pre-50S density maps from intermediate-containing fractions using cryo-EM. In the newly solved maps, we discovered the earliest intermediate ever reported, consisting of domain I at the 5’-end of 23S rRNA. To probe the mechanism behind the maps during assembly, we developed a novel density map segmentation and dependency analysis method. Ten cooperative assembly blocks were identified from segmentation of the maps, and these were organized into a block dependency map. This is the first time the dependencies on folding of rRNA helices and ribosomal protein binding could be integrated into a complete assembly map. In addition, we showed how the exit tunnel is folded on the solvent side, serving as a scaffold for 50S maturation. Using this new segmentation analysis method, we revisited previously reported bL17-depletion and ΔsrmB datasets. Most remarkably, the other two datasets are also consistent with the block dependency, implying a unified early assembly pathway and flexible maturation landscape in early 50S biogenesis.