Inhibition of <i>in vitro</i> protein synthesis by ppGpp

Legault, Lucienne; Jeantet, Claude; Gros, François

doi:10.1016/0014-5793(72)80412-5

Cited by 43 publications

(21 citation statements)

References 19 publications

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“…Previous studies have shown that ppGpp regulates translation in bacterial cells Cashel 1973, 1974;Legault et al 1972;Yoshida et al 1972). More recent reports have also indicated that ppGpp acts as an inhibitor of GTPase activity by binding to bacterial IF2 or EF-G (Milon et al 2006;Mitkevich et al 2010).…”

Section: Discussionmentioning

confidence: 98%

“…The bacterial global regulator ppGpp has also been suggested to regulate the translation apparatus in chloroplasts (Tozawa and Nomura 2011;Cashel 1973, 1974;Legault et al 1972;Yoshida et al 1972). To clarify the role of ppGpp in chloroplast translation, we examined its effect in the in vitro translation system based on pea chloroplast extract.…”

Section: Inhibitory Effect Of Ppgpp On Translation Activity Of the Chmentioning

confidence: 99%

“…Given that the structural features of ppGpp suggest that it acts as a competitor for GTP or GDP in the regulation of cellular functions in prokaryotes (Cashel et al 1996;Masuda et al 2008b) and that translation elongation factors are thought to be targets of ppGpp control in bacteria (Hamel and Cashel 1973;Legault et al 1972), we next investigated the effect of ppGpp on the peptide elongation cycle of translation in the pea chloroplast extract. In the presence of 0.5 mM ATP, the concentration of GTP in the range 0.2-0.8 mM did not affect polyphenylalanine synthesis activity (Fig.…”

Section: Peptide Elongation Activity In the Chloroplast Extractmentioning

confidence: 99%

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ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Nomura

Takabayashi

Kuroda³

et al. 2011

Plant Mol Biol

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Chloroplasts possess common biosynthetic pathways for generating guanosine 3',5'-(bis)pyrophosphate (ppGpp) from GDP and ATP by RelA-SpoT homolog enzymes. To date, several hypothetical targets of ppGpp in chloroplasts have been suggested, but they remain largely unverified. In this study, we have investigated effects of ppGpp on translation apparatus in chloroplasts by developing in vitro protein synthesis system based on an extract of chloroplasts isolated from pea (Pisum sativum). The chloroplast extracts showed stable protein synthesis activity in vitro, and the activity was sensitive to various types of antibiotics. We have demonstrated that ppGpp inhibits the activity of chloroplast translation in dose-effective manner, as does the toxic nonhydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate (GDPNP). We further examined polyuridylic acid-directed polyphenylalanine synthesis as a measure of peptide elongation activity in the pea chloroplast extract. Both ppGpp and GDPNP as well as antibiotics, fusidic acid and thiostrepton, inhibited the peptide elongation cycle of the translation system, but GDP in the similar range of the tested ppGpp concentration did not affect the activity. Our results thus show that ppGpp directly affect the translation system of chloroplasts, as they do that of bacteria. We suggest that the role of the ppGpp signaling system in translation in bacteria is conserved in the translation system of chloroplasts.

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Section: Discussionmentioning

confidence: 98%

Section: Inhibitory Effect Of Ppgpp On Translation Activity Of the Chmentioning

confidence: 99%

Section: Peptide Elongation Activity In the Chloroplast Extractmentioning

confidence: 99%

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ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Nomura

Takabayashi

Kuroda³

et al. 2011

Plant Mol Biol

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“…ObgE has been implicated in DNA replication (60) and has also been shown to bind to the Gramnegative (p)ppGpp synthetase/hydrolase enzyme SpoT from E. coli (61). Similar to RsgA, RbgA, HflX, and Era, it has recently been shown that ObgE also has a role in 50S and 30S ribosomal subunit association, and that (p)ppGpp binding can enhance the association of ObgE to the 50S subunit of the ribosome (59).…”

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.

188

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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“…Although ppGpp has been reported to inhibit the AUG-or R17 RNA-directed ribosomal binding of fMet-tRNAf (18), this compound has also been shown to interfere with the activity of the E. coli elongation factor EF-Tu (19) or that of the wheat-germ elongation factor EF-1 (20), thereby blocking the poly(U)-dependent ribosomal binding of Phe-tRNA mediated by these factors. However, the inhibitor reported in this paper has no effect on reactions mediated by the chain elongation factors EF-Tu and EF-Ts.…”

Section: Discussionmentioning

confidence: 99%

A Specific Inhibitor of Polypeptide-Chain Initiation in Escherichia coli

Lee‐Huang

Lee

Ochoa

1973

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

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An inhibitor of polypeptide-chain initiation was isolated from E. coli cells. This protein inhibits formation of the 30S or 70S initiation complex with either fMet-tRNAf as initiator and AUG, MS2 RNA, or late T4 RNA as messenger, or acPhe-tRNA as initiator and poly-(U) as messenger. Chain elongation, e.g., poly(U) translation at high Mg2+ concentration, is not inhibited. The inhibitor is rendered ineffective when active aminoacylation of tRNA is taking place, e.g., during natural mRNA translation. This inhibitor is distinct from the so-called interference (i) factors, which interfere exclusively with the action of initiation factor 3. Since the new inhibitor can apparently be turned on and off, it may have a regulatory function in translation.Revel and collaborators (1) isolated from 1.0 M NH4Cl washes of Escherichia coli ribosomes a protein factor that inhibits the initiation factor (IF)-3-dependent translationof MS2 RNAbut not that of late T4 RNA. They refer to this protein as the i (interference) factor. This factor was identified (2, 3) as the a subunit of Q0 replicase, one of the three subunits contributed by the host cell (4, 5). We have described (6, 7) the isolation of two messenger-discriminating species of E. coli IF-3, referred to as 1F-3a and IF-33. IF-3a has selectivity for such messengers as MS2, E. coli, and early T4 RNA, whereas IF-3,3 selects predominantly for late T4 RNA. While Revel's i factor (ia) is specific for IF-3a, we found one other factor (id) specific for IF-3,3 in the high-salt ribosomal wash (8).We now report on the isolation of a new inhibitor which, unlike the i factors, inhibits formation of the initiation complex in systems, e.g., AUG-dependent ribosomal binding of fMet-tRNAf, poly(U)-dependent ribosomal binding of acPhe-tRNA, that do not require IF-3. The inhibitor is rendered inactive under optimal conditions for natural mRNA (e.g., MS2 RNA) translation, when tRNA is being actively aminoacylated. The fact that the inhibitor may be either active or inactive suggests that it may play a role in regulation of translation. MATERIALS AND METHODSThe Inhibitor was first discovered in high-salt ribosomal washes. Later it was found also in the high-speed supernatant. An outline of its purification from the 1.0 M NH4C1 wash of E. coli MRE600 ribosomes is given here. Cells grown as described (6) were harvested at mid-logarithmic phase and frozen until used. Preparation and washing of the ribosomes Abbreviations: acPhe-tRNA, N-acetylphenylalanyl-transfer RNA; Mg(OAc)2, magnesium acetate; IF, initiation factor; i factor, interference factor; EF, elongation factor. * To whom reprint requests should be sent.was as described (7)

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Inhibition of in vitro protein synthesis by ppGpp

Cited by 43 publications

References 19 publications

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

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

A Specific Inhibitor of Polypeptide-Chain Initiation in Escherichia coli

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