Dataset Information

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

ABSTRACT: The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles-S3, S10, S14, and S21-all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increased levels of precursor 17S rRNA in assembly intermediates. Mutation ?lepA confers a synthetic growth phenotype in absence of RsgA, another GTPase, well known to act in 30S subunit assembly. Analysis of the ?rsgA strain reveals accumulation of intermediates that resemble those seen in the absence of LepA. These data suggest that RsgA and LepA play partially redundant roles to ensure efficient 30S assembly.

SUBMITTER: Gibbs MR

PROVIDER: S-EPMC5293072 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Gibbs Michelle R MR Moon Kyung-Mee KM Chen Menglin M Balakrishnan Rohan R Foster Leonard J LJ Fredrick Kurt K

Proceedings of the National Academy of Sciences of the United States of America 20170117 5

The physiological role of LepA, a paralog of EF-G found in all bacteria, has been a mystery for decades. Here, we show that LepA functions in ribosome biogenesis. In cells lacking LepA, immature 30S particles accumulate. Four proteins are specifically underrepresented in these particles-S3, S10, S14, and S21-all of which bind late in the assembly process and contribute to the folding of the 3' domain of 16S rRNA. Processing of 16S rRNA is also delayed in the mutant strain, as indicated by increa ...[more]

PMID: 28096346

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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

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

Publications

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

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