The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which is poorly understood. Although several bona fide assembly factors have been identified, their precise functions and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli GTPase, CgtA E , in late steps of large ribosomal subunit biogenesis. CgtA E belongs to the Obg/CgtA GTPase subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in CgtA E cause both polysome and rRNA processing defects; small-and large-subunit precursor rRNAs accumulate in a cgtA E mutant. In this study we apply a new semiquantitative proteomic approach to show that CgtA E is required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribosomal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonribosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships between these proteins and CgtA E . We also show that purified CgtA E associates with purified ribosomal particles in the GTP-bound form. Finally, CgtA E cofractionates with the mature 50S but not with intermediate particles accumulated in other large ribosome assembly mutants.Although assembly of prokaryotic ribosomes from purified ribosomal proteins (r-proteins) and rRNAs can occur independently in vitro (51, 52, 75), accumulating evidence suggests that, as in eukaryotes, in vivo prokaryotic ribosome biogenesis depends on the aid of nonribosomal factors. The higher temperature, increased Mg 2ϩ concentration, and longer incubation times necessary for in vitro relative to in vivo conditions (51) imply that the likely role of accessory factors is to expedite ribosome maturation by reducing the activation energies for the rate-limiting reactions. Although not complicated by the involvement of different cellular compartments, the prokaryotic ribosome assembly process is far from simple, requiring coordinated synthesis of 3 rRNAs (5S, 16S, and 23S) and 55 r-proteins, processing and modification of these components, and their appropriate sequential unification to produce mature ribosomes. The details of how this process is controlled temporally, even spatially, in the small bacterial cell are incompletely understood.More than 170 nonribosomal proteins that transiently associate with different preribosomal particles have been identified in Saccharomyces cerevisiae (19,22,38,62), largely due to progress in combining biochemical affinity purification methods with newly developed proteomic techniques (24,25,29,54,58,61). By contrast, only a few such assembly factors have been found in bacteria, and most were identified via conventional genetic methods. These proteins consist of RNA-modifying enzymes such as methyltransferases and pseudouridine synthases, RNA-remodeling proteins such as RNA helicases, chaperones, GTPases, and proteins with unknown functions (1,