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

质体基因的表达对植物发育至关重要，主要在转录后和翻译水平上受到调控。然而，关于陆地植物叶绿体的翻译调控，包括所涉及的机制和参与者，以及翻译和共翻译过程的耦合（如蛋白质靶向），我们知之甚少。该项目旨在更深入地了解叶绿体翻译行为、翻译调控以及参与这些过程的蛋白质。通过对翻译核糖体在mrna上留下的足迹进行深度测序，核糖体分析能够对翻译体进行全面分析。这提供了关于翻译核糖体的位置和丰度的全基因组信息，反映了翻译状态。最近，我修改了这种方法，用高分辨率平铺微阵列代替深度测序步骤，以节省时间和成本分析叶绿体翻译。微阵列和深度测序方法的结合将促进叶绿体翻译的分析。基于快速阵列的方法将分别用于不同发育阶段和叶绿体基因表达和光合能力改变的突变体的翻译动力学的初步分析。随后，深度测序方法将允许对选定的缺陷和影响进行更详细的研究。此外，我最近调整了该方法，以方便检查叶绿体翻译的亚细胞器定位的空间动力学。再加上鉴定翻译因子及其功能表征的创新核糖学方法，这将使我能够解决以下关于模式植物烟草叶绿体翻译的问题：(i)叶绿体翻译组是如何定义的（orf，启动和停止密码子，剪接位点，编辑位点等）？（ii）在植物发育过程中，翻译调控在多大程度上影响叶绿体蛋白质组的动态？（iii）在陆地植物叶绿体中，装配依赖的反馈调节是否控制着光合复合体亚基的表达，就像我们已知的绿藻衣藻中发生的那样？（四）调节翻译的顺式因素和反式因素有哪些？

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?