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ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro

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Abstract

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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Acknowledgments

We thank Professor M. Sugiura (Nagoya University) for critical comments, R. Uno for technical assistance and members of the Cell-Free Science and Technology Research Center of Ehime University for many helpful discussions. This work was supported by a grant-in-aid for Scientific Research (no. 21570047 to Y.T.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Authors and Affiliations

  1. Division of Biomolecular Engineering, Cell-Free Science and Technology Research Center, and Venture Business Laboratory, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan

    Yuhta Nomura, Taito Takabayashi, Akira Nozawa & Yuzuru Tozawa

  2. Graduate School of Natural Sciences, Nagoya City University, Nagoya, 467-8501, Japan

    Hiroshi Kuroda & Yasushi Yukawa

  3. The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, 790-8566, Japan

    Kwanchanok Sattasuk & Mitsuru Akita

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  1. Yuhta Nomura
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Correspondence to Yuzuru Tozawa.

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Nomura, Y., Takabayashi, T., Kuroda, H. et al. ppGpp inhibits peptide elongation cycle of chloroplast translation system in vitro. Plant Mol Biol 78, 185–196 (2012). https://doi.org/10.1007/s11103-011-9858-x

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