PTC Remodeling in Pre50S Intermediates: Insights into the Role of DEAD-box RNA Helicase DbpA

Abstract

Ribosome biogenesis is a complex and fundamental multi-step process in vivo, requiring numerous assembly factors to facilitate and regulate the subunit assembly. The Escherichia coli DEAD-box RNA helicase, DbpA, is known to promote the assembly of the peptidyl transferase center (PTC) during 50S subunit maturation, specifically through the binding of the H92 hairpin in 23S rRNA. In this study, we employed cryoelectron microscope to characterize a series of pre50S snapshots isolated from an E.coli strain overexpressing a DbpA(R331A) mutant in the background of depleting dbpA gene. Our structural analysis defined a collection of late-stage particles along a progressive assembly pathway, revealing intermediates that were immature in the PTC, with domain V undergoing large conformational changes and progressive folding. In the pre50S intermediates, we observed mismatched base-pairings in H73-H74, which define the possible unwinding substrate of DbpA. Furthermore, the structures identified RsfS and YjgA as participating in 50S maturation with DbpA, and label-free quantitive mass spectrum revealed the involvement of multiple assembly factors and rRNA modification enzymes in these intermediates. In summary, our study provides mechanistic insights into how the DbpA, in concert with other assembly factors, facilitates the maturation of the 50S subunit in prokaryotes.