Protein biosynthesis in chloroplasts: defining the translatome and elucidating mechanisms of translational regulation

点击翻译按钮获取中文摘要

Plastid gene expression is essential for plant development and mainly regulated at the post-transcriptional and translational levels. However, little is known about translational regulation in land plant chloroplasts including the mechanisms and players involved, and the coupling of translational and co-translational processes (e.g. protein targeting). This project aims at a deeper understanding of chloroplast translation behavior, translational regulation, and the proteins that participate in these processes. Ribosome profiling enables the comprehensive analysis of translatomes by deep sequencing of footprints that translating ribosomes leave on mRNAs. This provides genome-wide information about position and abundance of translating ribosomes, reflecting the translational state. Recently, I have modified this method for the time and cost efficient analysis of chloroplast translation by substituting the deep-sequencing step by high-resolution tiling microarrays. Combined use of both microarray and deep sequencing approaches will boost the analysis of chloroplast translation. The rapid array-based method will be used for initial analyses of translational dynamics in different developmental stages and mutants with altered chloroplast gene expression and photosynthesis capacity, respectively. Subsequently, the deep sequencing method will allow the more detailed study of selected defects and effects. Additionally, I recently adapted the approach to facilitate the examination of spatial dynamics in the suborganellar localization of chloroplast translation. Complemented by innovative ribonomic approaches for the identification of translation factors and their functional characterization, this will allow me to address the following questions about chloroplast translation in the model plant tobacco:(i) How is the chloroplast translatome defined (ORFs, start and stop codons, splice sites, editing sites, etc.)?(ii) To what extent does translational regulation contribute to the dynamics in the chloroplast proteome during plant development?(iii) Do assembly-dependent feedback regulations control the expression of subunits of photosynthetic complexes in land plant chloroplasts as it is known to occur in the green alga Chlamydomonas?(iv) What are the cis-elements and trans-factors that regulate translation?