Three bacterial GTPases—YihA, EngA, and ObgE—act as successive placeholders that coordinate the temporal maturation of distinct functional regions within the large ribosomal subunit, while EngA and BipA additionally function as regulatory translation factors that bridge ribosome assembly to translation initiation, establishing a GTPase-mediated surveillance system that monitors both ribosome biogenesis and translational fidelity.

Ribosome biogenesis and mRNA translation are fundamental cellular processes regulated by a diverse set of protein factors, including GTPases. In bacteria, while several GTPases are known to participate in ribosome assembly, their precise mechanisms in subunit maturation and their potential roles in translation regulation remain largely elusive. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of native pre-50S assembly intermediates and 70S translating ribosomes, isolated via epitope-tagged GTPases from engineered Escherichia coli cells at resolutions of 2.3-4.4 Å. These structures elucidate how three GTPases, YihA, EngA and ObgE, act as successive placeholders to mediate rRNA folding and to coordinate the correct timing of the maturation of different functional blocks within the large ribosomal subunit. Furthermore, our data identify several previously unrecognized 70S translational complexes bound by the GTPases EngA and BipA—factors traditionally regarded as assembly factors, thereby uncovering their regulatory role in bridging ribosome assembly and translation initiation. Collectively, our findings delineate a GTPase-mediated surveillance system that continuously monitors the assembly of ribosomal subunits and translation adversity, thereby safeguarding protein synthesis and maintaining proteome homeostasis.