Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.

Identification of a ribosomal ATPase in Escherichia coli cells.

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.