Biochemical Characterization of Ribosome Assembly GTPase RbgA in Bacillus subtilis

Achila, David; Gulati, Megha; Jain, Nikhil; Britton, Robert A.

doi:10.1074/jbc.m111.331322

Cited by 41 publications

(78 citation statements)

References 32 publications

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“…In normal unstressed cells, RbgA and HflX bind to the 50S subunit (40,42,48,49), whereas both RsgA and Era bind to the 30S subunit and interact with the 16S rRNA (35,36,43,44). There is evidence to suggest that this occurs while the proteins are in the GTP-bound state, as for RsgA, RbgA, and ObgE, the inhibition of GTPase activity by the binding of nonhydrolysable analog of GTP causes increased association of the protein to ribosomal subunits (36,45,64). Here, the proteins are thought to have a caretaking or checkpoint role where they could function to facilitate proper RNA folding or processing or could promote correct protein-protein or protein-RNA interactions.…”

Section: Discussionmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

185

227

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The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Grampositive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Grampositive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases-RsgA, RbgA, Era, HflX, and ObgE-as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria.ribosome | stringent response | tolerance | ppGpp | Staphylococcus aureus

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Section: Discussionmentioning

confidence: 99%

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Corrigan

Bellows

Wood

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

185

227

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“…RbgA in B. subtilis is essential for cellular growth and participates in the late steps of 50S LSU ribosome assembly and maturation. It interacts with the 50S subunit in a GTP-dependent manner and its GTPase activity induces its dissociation from the ribosome [90]. According to in vitro studies the GTPase activity is stimulated more than 60-fold in the presence of the 50S subunit [87,[89][90][91].…”

Section: 21-gtpasesmentioning

confidence: 99%

“…It interacts with the 50S subunit in a GTP-dependent manner and its GTPase activity induces its dissociation from the ribosome [90]. According to in vitro studies the GTPase activity is stimulated more than 60-fold in the presence of the 50S subunit [87,[89][90][91]. In RbgA depleted cells the mature 50S subunits are not formed; instead, a 45S complex (lacking ribosomal proteins -L16 and L36 [86,[90][91][92][93][94], and reduced amounts of L27, L33, and L35 [92]) is accumulated.…”

Section: 21-gtpasesmentioning

confidence: 99%

“…According to in vitro studies the GTPase activity is stimulated more than 60-fold in the presence of the 50S subunit [87,[89][90][91]. In RbgA depleted cells the mature 50S subunits are not formed; instead, a 45S complex (lacking ribosomal proteins -L16 and L36 [86,[90][91][92][93][94], and reduced amounts of L27, L33, and L35 [92]) is accumulated. Spontaneous rbgA suppressors have been mapped to the gene encoding L6, and in vitro maturation assays have suggested that RbgA is necessary for correct positioning of L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins [92].…”

Section: 21-gtpasesmentioning

confidence: 99%

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Mitochondrial ribosome assembly in health and disease

et al. 2015

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The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

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“…His 6 -CpgA Association with 30 and 50 S Subunits (Co-sedimentation Assays)-A mixture of 21 pmol of B. subtilis 70 S ribosome (A 260 ϭ 0.9) and 84 pmol of His 6 -CpgA was preincubated for 15 min in the presence of 100 M of the nonhydrolyzable GTP analog GMP-PNP 3 that was shown to strongly enhance the interaction between GTPases and ribosomal subunits; in particular it stimulates the interaction between RsgA and the 30 S ribosomal subunit (1,24,30). Then the sample was sedimented through a 5 to 20% (w/v) sucrose gradient in standard buffer (10 mM Tris-HCl, pH 7.5, 0.1 mM magnesium acetate, 30 mM KCl) at 200,000 ϫ g at 4°C for 190 min in a SW41Ti rotor (Beckman).…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation of CpgA Protein Enhances Both Its GTPase Activity and Its Affinity for Ribosome and Is Crucial for Bacillus subtilis Growth and Morphology

Pompeo¹,

Freton²,

Wicker‐Planquart³

et al. 2012

Journal of Biological Chemistry

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Background:CpgA is an essential GTPase phosphorylated in vitro by the Ser/Thr kinase PrkC. Results: CpgA is phosphorylated on Thr-166, and phosphomimetic or phosphoablative replacements of this residue in CpgA modify its GTPase activity. Conclusion: Phosphorylation of CpgA is probably a key regulatory feature of B. subtilis physiology. Significance: The regulation of CpgA via phosphorylation may be needed in sporulating B. subtilis cells.

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Biochemical Characterization of Ribosome Assembly GTPase RbgA in Bacillus subtilis

Cited by 41 publications

References 32 publications

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

Mitochondrial ribosome assembly in health and disease

Phosphorylation of CpgA Protein Enhances Both Its GTPase Activity and Its Affinity for Ribosome and Is Crucial for Bacillus subtilis Growth and Morphology

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