Translation and its regulation in different compartments of the plant cell

• Applicant: Professor Dr. Christian M. T. Spahn
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 416210002

Because plants are not mobile, they have acquired special adaptation mechanisms during evolution to adapt to changing environmental conditions. One of the most fundamental adaptation processes is the rapid regulation of gene expression, which in plants is highly modulated at the level of mRNA translation. Structural and mechanistic studies of protein biosynthesis have so far focused heavily on bacteria, lower eukaryotes (yeast) and mammals, whereas our knowledge of translation in plants is limited. In plants, translation occurs in three different cellular compartments: cytosol, chloroplast and mitochondrion. Each of these cell compartments has a specialized translation apparatus. The aim of this proposal is to elaborate the structural basis of protein synthesis and its regulation and adaptation to environmental conditions in plant cells in two different compartments, namely the cytosol and the chloroplast. To this end, we propose a two-pronged strategy. Continuing our successful work from the first funding period, we will analyze native, actively translating, ribosomal complexes (ex vivo polysomes) from the cell using single-particle cryo-EM. This will allow us to solve the structures of the longer-lived elongation intermediates at very high resolution (<4 Å - ~2 Å) and to conduct structural analysis down to the level of chemical modifications of the ribosomal RNAs and proteins as well as bound cations, polyamines and water molecules. To visualize translation in the native cellular context of the cytosol or chloroplasts, we will use cryo-electron tomography in conjunction with subtomogram averaging. Using in situ analysis, we will obtain a comprehensive overview of the translation intermediates including the transient decoding and translation complexes with the translational GTPases eEF1A/EF-Tu and eEF2/EF-G. In addition, the distribution of functional states will determine the energy landscape of translation in the native cellular context. To gain insight into the molecular mechanisms underlying a plant's response to cold adaptation or cold stress, we will extend our analysis to plants grown at low temperature. Our preliminary analysis has already shown that exposure of plants to cold stress leads to significant changes in the translational energy landscape underlying the ribosomal elongation cycle in both the chloroplast and the cytosol. Furthermore, we will investigate the role of the ribosomal proteins bL33c and uS15c in chloroplast translation, with a focus on their relevance in cold acclimation.

• DFG Programme: Research Grants