Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

Mitkevich, Vladimir A.; Ermakov, Andrey; Kulikova, Alexandra A.; Tankov, Stoyan; Shyp, Viktoriya; Soosaar, Andres; Tenson, Tanel; Makarov, Alexander А.; Ehrenberg, Måns; Hauryliuk, Vasili

doi:10.1016/j.jmb.2010.08.016

Cited by 78 publications

(76 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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). We observed that ppGpp remained largely stable during inhibition of translation activity in the pea chloroplast extract, likely accounting for the clear concentration-dependent effect of ppGpp on the time course of peptide elongation.…”

Section: Discussionsupporting

confidence: 63%

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Nomura

Takabayashi

Kuroda³

et al. 2011

Plant Mol Biol

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 63%

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Nomura

Takabayashi

Kuroda³

et al. 2011

Plant Mol Biol

View full text Add to dashboard Cite

show abstract

“…Thermodynamic analysis revealed that ppGpp binds to IF2 with higher affinity than to EF-G. Binding of fMet-tRNA fMet to IF2 occurs with little variation in the presence of ppGpp compared to GTP while it is very sensitive to the nucleotides when complexed with the 30S subunit [127]. These results support the notion that initiation of translation is preferentially regulated by ppGpp under conditions of nutrient starvation.…”

Section: Targets For Control Of Translationsupporting

confidence: 63%

Protein Synthesis and the Stress Response

Katz¹,

Orellana²

2012

Cell-Free Protein Synthesis

View full text Add to dashboard Cite

“…The (p)ppGpp alarmone exerts its regulatory role chiefly via direct binding to the bacterial RNA polymerase (3), provoking changes in the profile of transcribed messages. (p)ppGpp also regulates bacterial replication via binding to DNA primase (4) and inhibits translation initiation via binding to initiation factor IF2, a translational GTPase (5,6). Evidently, the stringent response is a core cellular adaptation pathway, acting as a hub in the regulatory network, where it integrates information about the nutritional status of the bacterial cell and regulates cellular metabolism on the transcription, translation, and replication levels.…”

mentioning

confidence: 99%

Single-molecule investigations of the stringent response machinery in living bacterial cells

English

Hauryliuk

Sanamrad

et al. 2011

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

Self Cite

261

294

View full text Add to dashboard Cite

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism. cytosolic diffusion | single particle tracking | photoactivated localization microscopy | stroboscopic illumination

show abstract

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

Cited by 78 publications

References 40 publications

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

Protein Synthesis and the Stress Response

Single-molecule investigations of the stringent response machinery in living bacterial cells

Contact Info

Product

Resources

About