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Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation.

ABSTRACT: Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying out guanosine triphosphate (GTP) hydrolysis. To probe RbgA function in ribosome assembly, we used a combined bioinformatics, genetic and biochemical approach. We identified a RNA-binding domain within the C-terminus of RbgA that is structurally similar to AmiR-NasR Transcription Anti-termination Regulator (ANTAR) domains, which are known to bind structured RNA. Mutation of key residues in the ANTAR domain altered RbgA association with the ribosome. We identified a putative catalytic residue within a highly conserved region of RbgA, His9, which is contained within a similar PGH motif found in elongation factor Tu (EF-Tu) that is required for GTP hydrolysis on interaction with the ribosome. Finally, our results support a model in which the GTPase activity of RbgA directly participates in the maturation of the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.

SUBMITTER: Gulati M

PROVIDER: S-EPMC3597669 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation.

Gulati Megha M Jain Nikhil N Anand Baskaran B Prakash Balaji B Britton Robert A RA

Nucleic acids research 20130115 5

Ribosome biogenesis GTPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying o ...[more]

PMID: 23325847

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Project description:BackgroundYqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.Methodology/principal findingsMnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only approximately 25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix alpha2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.Conclusions/significanceAn uncommon means to achieve GTP hydrolysis utilizing a K(+) ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K(+) driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases.

Dataset Information

Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation.

Publications

Mutational analysis of the ribosome assembly GTPase RbgA provides insight into ribosome interaction and ribosome-stimulated GTPase activation.

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