TheEscherichia coliGTPase CgtA_EIs Involved in Late Steps of Large Ribosome Assembly

Abstract

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,

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…Based on these and additional data, the authors propose that the final stages of ribosome maturation take place in conjunction with its first round of initiation. This model can rationalize several earlier observations, including the presence of precursor rRNAs in polysomes (36,37), links between IF2 and ribosome assembly (12,38), and inhibition of 16S rRNA processing resulting from defects in 50S assembly (39,40).…”

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

“…1C) (15) or ObgE (16). It remains unknown, however, whether this 45S particle is able to form an active 50S subunit (21).…”

“…Overexpression of the R331A active-site mutant of DbpA in Escherichia coli results in accumulation of a particle that sediments at 45S at low Mg 2+ concentration, but increases in density and comigrates with the 50S subunit at high Mg 2+ concentration (15), suggesting that the 45S particle is structurally flexible and can undergo conformational changes upon binding of Mg 2+ . A similar 45S particle that undergoes Mg 2+ -dependent alteration in density appears in cells expressing the G80E/D85N mutant of ObgE (CgtA E ) (16), an essential GTPase involved in the late steps of 50S assembly (10).…”

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae

“…In E. coli, CgtA is involved in late steps of ribosome assembly and maturation (24,25), and is specifically required for optimal incorporation of specific ribosomal proteins into the large ribosomal subunit (26). This is probably a conserved function because in other organisms, such as V. harveyi, B. subtilis, C. crescentus, and E. coli, CgtA interacts with the 50S ribosomal subunit and has been shown to be involved in assembly or stability of the 50S subunit (17,23,27,28).…”