Biochemical characterization of ribosome assembly GTPase RbgA in Bacillus subtilis

Abstract

The ribosome biogenesis GTPase A protein RbgA is involved in the assembly of the large ribosomal subunit in Bacillus subtilis, and homologs of RbgA are implicated in the biogenesis of mitochondrial, chloroplast, and cytoplasmic ribosomes in archaea and eukaryotes. The precise function of how RbgA contributes to ribosome assembly is not understood. Defects in RbgA give rise to a large ribosomal subunit that is immature and migrates at 45 S in sucrose density gradients. Here, we report a detailed biochemical analysis of RbgA and its interaction with the ribosome. We found that RbgA, like most other GTPases, exhibits a very slow k(cat) (14 h(-1)) and has a high K(m) (90 μM). Homology modeling of the RbgA switch I region using the K-loop GTPase MnmE as a template suggested that RbgA requires K(+) ions for GTPase activity, which was confirmed experimentally. Interaction with 50 S subunits, but not 45 S intermediates, increased GTPase activity by ∼55-fold. Stable association with 50 S subunits and 45 S intermediates was nucleotide-dependent, and GDP did not support strong interaction with either of the subunits. GTP and guanosine 5'-(β,γ-imido)triphosphate (GMPPNP) were sufficient to promote association with the 45 S intermediate, whereas only GMPPNP was able to support binding to the 50 S subunit, presumably due to the stimulation of GTP hydrolysis. These results support a model in which RbgA promotes a late step in ribosome biogenesis and that one role of GTP hydrolysis is to stimulate dissociation of RbgA from the ribosome.

FIGURE 1.

Kinetic analysis of GTP hydrolysis rates of RbgA proteins. GTP hydrolysis rates of RbgA proteins were determined by monitoring the release of free phosphate using the malachite green-ammonium molybdate colorimetric assay as described “Experimental Procedures.” Reactions were carried out under initial rate conditions (<10% of substrate consumed). Values of k_cat and K_m were determined by fitting the Michaelis-Menten equation using nonlinear regression algorithms provided by the GraphPad Prism software. Representative GTP hydrolysis curves for wild-type RbgA (●) and the P-loop variant S134A (■) are shown. Error bars represent S.D. of three technical replicates.

FIGURE 2.

Stimulation of GTPase activity of RbgA by ribosomal particles. Stimulation of RbgA GTPase activity by ribosomes was assessed by determining the GTP hydrolysis rates of RbgA in the presence of various purified ribosomal particles. 100 nm purified ribosome was incubated with 100 nm RbgA for 15 min at 37 °C in the presence of various concentrations of GTP and assayed as described under “Experimental Procedures.” The representative curves are of GTP hydrolysis rates determined based on the free phosphate produced after the reactions had proceeded for 15 min at 37 °C with reaction mixtures containing RbgA only (●), RbgA and the mature 50 S subunit (■), RbgA and the free 50 S subunit (▴), and RbgA and the 45 S intermediate (▾). Error bars represent S.D. of three technical replicates.

FIGURE 3.

Superimposition of MnmE and homology model of RbgA. A, an MnmE and RbgA homology model was superimposed using the LigAlign script in PyMOL, which aligned the two structures based on their ligand positions, which is GDP in this case. The K-loops of MnmE (green) and RbgA (brown) occupy a similar position (boxed) around the bound potassium in the crystal structure. B, an enlarged view of the catalytic pocket with GDP and bound potassium is shown in which Asn-130 from the P-loop of RbgA (brown) coordinates the bound K⁺ ion (indicated by a purple sphere) in a similar fashion as Asn-226 from the P-loop of MnmE (green) (indicated by arrows).

FIGURE 4.

Interaction between RbgA and ribosome in presence of different guanine nucleotides. Binding of RbgA to purified ribosomal subunits was tested by an in vitro binding assay in which 60 pmol of RbgA was preincubated with 1.5 mm GDP, GTP, GMPPNP, or pppGpp before the addition of 10 pmol of purified 45 S or 50 S ribosomal subunits. The mixtures were incubated for an additional 15 min at 37 °C, and free RbgA was filtered off by centrifugation through a 100-kDa cutoff Microcon column. The columns were washed three times, first with buffer B and then twice with buffer C (high salt buffer). RbgA-ribosome complexes were eluted, and bound RbgA was detected by immunoblotting using anti-RbgA antibody.

FIGURE 5.

Proposed model for role of RbgA in 50 S subunit maturation. RbgA interacts with the 45 S intermediate in the GTP-bound form and introduces conformational changes that further facilitate the binding of late ribosomal proteins such as L16, L27, and L36 (depicted by ovals). Binding of these proteins promotes maturation of the 45 S intermediate (white) to a mature 50 S subunit (gray). Along with the complete maturation of the 50 S subunit, GTP at RbgA is hydrolyzed to GDP, and this GDP-bound RbgA and inorganic phosphate leave the mature 50 S subunit, which is now ready to take part in translation. It is also possible that RbgA completes maturation of a late step that does not end in the final maturation of the 50 S subunit.