Functional proteomics of chloroplast nucleoproteins towards an understanding of nucleoid structure, function and dynamics

叶绿体类核含有一组DNA结合蛋白，其中一些具有结构功能，而另一些可能作为基因表达的关键调节因子。我们建议利用模式生物莱茵衣藻(又称“绿色酵母”)的巨大遗传潜力来解决叶绿体类核结构和功能的关键问题。这项研究包括大规模的衣藻叶绿体类核的纯化和系统的蛋白质组学分析，最终目的是确定与叶绿体基因组相关的完整的DNA结合蛋白(结构类核蛋白和调节蛋白)。我们的目标是从分子上表征这些因子的一个子集，此外，我们还将通过比较分析与不同环境条件和发育阶段的叶绿体类核相关的一组DNA结合蛋白来探索类核结构的动力学。这种比较功能蛋白质组学方法将为调控叶绿体DNA的高阶结构以及通过DNA结合调节蛋白调控叶绿体基因表达以响应不断变化的环境条件和发育程序提供新的见解。审查小组的意见(包括一份书面评论)：博克教授希望从衣藻叶绿体中分离出类核物质，并鉴定这些结构中包含的蛋白质。然后，应检查这些蛋白质是否为GFP融合，以确定其在类核中的假定定位。本项目的最终目的是通过RNAi技术分析类核蛋白的功能，并进一步比较不同条件下类核蛋白的蛋白质组成。博克教授在高等植物叶绿体遗传学和分子生物学方面经验丰富，在这一领域有出色的出版记录。最初的书面建议中概述的项目被认为非常有趣和及时，因为人们对叶绿体类核蛋白知之甚少，而且对理解原核类核的预期影响也是如此。然而，评审者们担心，这一设置过于宽泛，无法让该项目超出对大量候选蛋白质的编目范围。因此，他们要求对蛋白质给出明确的选择标准，以便在功能层面上进一步确定其特征，从而更严格地关注该项目。此外，还要求澄清一些技术细节。

Chloroplast nucleoids contain a set of DNA-binding proteins some of which have architectural function whereas others are likely to act as key regulators of gene expression. We propose to harness the enormous genetic potential of the model organism Chlamydomonas reinhardtii (also referred to as "green yeast") in order to address key questions of chloroplast nucleoid structure and function. The proposed research involves the large-scale purification of chloroplast nucleoids from Chlamydomonas and their systematic proteomics analysis with the ultimate goal to identify the complete set of DNA-binding proteins (structural nucleoid proteins and regulator proteins) associated with the chloroplast genome. We aim to characterize a subset of these factors molecularly and, in addition, we will explore the dynamics of nucleoid architecture by comparatively analyzing the set of DNA-binding proteins associated with chloroplast nucleoids in various environmental conditions and developmental stages. This comparative functional proteomics approach will provide new insights into the regulation of higher-order structures of the chloroplast DNA and the control of chloroplast gene expression through DNA-binding regulator proteins in response to changing environmental conditions and developmental programs. Opinion of the review panel (including one written comment): Prof. Bock wants to isolate nucleoids from Chlamydomonas chloroplasts and identify the proteins contained in these structures. The proteins shall then be checked as GFP fusions for their presumed localisation within the nucleoids. The final aim of this project is to analyse the function of nucleoid proteins by means of the RNAi technology and, furthermore, by comparing the protein composition of nucleoids under different conditions. Prof. Bock is well experienced in genetics and molecular biology of higher plant chloroplasts and has an excellent publication record in this field. The project outlined in the original written proposal was considered very interesting and timely because little is yet known about chloroplast nucleoid proteins and because of the expected impact on understanding prokaryotic nucleoids. However, the reviewers were worried that the set-up was too broad to allow the project to proceed much beyond cataloguing of a large number of candidate proteins. They, therefore, requested that clear selection criteria be given for proteins to be characterized further on a functional level, and thereby the project be more strictly focussed. In addition, a few technical details were asked to be clarified.