Defining the molecular basis of chloroplast transcription of photosynthetic genes

植物生长是由光合作用驱动的。然而，植物如何产生光合作用蛋白质还不清楚。叶绿体包含编码关键光合蛋白的基因组和表达它们的独特分子机制。尽管叶绿体基因表达机制非常重要，但其如何发挥作用还没有被详细描述。叶绿体基因产生光合作用蛋白的第一步是转录产生信使RNA（mRNA）。这一过程是由一个大的蛋白质组装，称为质体编码的聚合酶（PEP）。由于PEP的转录活性的激活，植物响应于光而变绿色。此外，植物胁迫如干旱、高温和病原体攻击影响PEP活性，以允许编码光合蛋白的特定基因被打开或关闭。尽管PEP在植物发育和适应中起着重要作用，但人们对PEP如何转录叶绿体基因知之甚少。PEP由19种不同的蛋白质亚基组成，每种亚基都有重要的作用。PEP在转录酶中是显著的，因为它包含两个进化起源的亚基。核心类似于细菌的酶，并从蓝藻祖先的叶绿体基因组遗传。相比之下，12种或更多稳定结合到核心的蛋白质在核基因组中编码。因此，我们期望这些蛋白质，称为PAP（PEP相关蛋白），协调叶绿体独特的关键调控过程。为了更好地了解光合蛋白是如何由植物产生的，我们的目标是可视化PEP转录基因。为此，我们将使用低温电子显微镜（cryo-EM）收集PEP分子的图像。通过处理这些图像，可以构建原子分辨率的PEP模型。这些有望显示PAP如何激活叶绿体转录。现代冷冻EM提供的细节水平对于开发新的假设非常有价值，因为可以通过可预测的活动变化来设计精确的修改。在本项目中，我们还将研究使用纯化成分重建的转录反应和植物遗传互补实验进行特定变化的后果。结果将是更好地了解PEP的每个组成部分有什么作用，它如何执行它，以及为什么这些过程是叶绿体发育和光合作用所必需的。该项目预计将加深我们对转录的生化基础的基本理解。对真核细胞核和细菌中进行转录的蛋白质已经进行了数十年的详细研究。这表明，整理关于不同蛋白质的信息对于推断基因表达如何调控的一般原则至关重要。因此，了解作用于叶绿体基因的独特蛋白质组代表了一个令人兴奋的机会。转录调控是人类健康和疾病的关键组成部分，因此这项研究具有多种潜在用途。光合作用在产生维持地球上大部分生命的氧气和能量方面发挥着核心作用。对光合作用蛋白的详细结构和生化研究详细揭示了它们如何利用太阳能，这为作物改良和多种生物技术的发展提供了宝贵的基础。相比之下，相当的基因表达过程的机制研究，支持生产的光合作用蛋白在很大程度上缺乏。该项目将回答一系列互补的问题：是什么决定了光合作用蛋白质生产的时间和水平，以及我们如何修改它以开发更健壮的作物和新的生物技术应用？

Plant growth is driven by photosynthesis. However, it is not well understood how plants produce their photosynthetic proteins. The chloroplast contains a genome that encodes key photosynthetic proteins and a unique molecular machinery that expresses them. Despite their importance, how the chloroplast gene expression machinery functions has not been characterised in detail.The first stage in the production of photosynthetic proteins from chloroplast genes is their transcription to produce messenger RNAs (mRNAs). This process is performed by a large assembly of proteins known as the plastid-encoded polymerase (PEP). Plants turn green in response to light due to the activation of the transcriptional activity of PEP. In addition, plant stresses such as drought, heat and pathogen attack affect PEP activity to allow specific genes encoding photosynthetic proteins to be turned on or off. Despite its central role in plant development and adaptation, how PEP transcribes chloroplast genes is poorly understood. PEP is made of 19 different protein subunits that each have an essential role. PEP is remarkable amongst transcription enzymes in that it contains subunits of two evolutionary origins. The core resembles bacterial enzymes and was inherited with the chloroplast genome from a cyanobacterial ancestor. By contrast, the twelve or more proteins that stably bind to the core are encoded in the nuclear genome. We therefore expect that these proteins, known as PAPs (PEP-associated proteins), orchestrate key regulatory processes unique to the chloroplast.To better understand how photosynthetic proteins are produced by plants, we aim to visualise PEP as it transcribes genes. To do this, we will collect images of PEP molecules using cryogenic electron microscopy (cryo-EM). By processing these images, models of PEP at atomic resolution can be constructed. These are expected to show how PAPs activate chloroplast transcription. The level of detail provided by modern cryo-EM is immensely valuable to developing new hypotheses, as precise modifications can be designed with predictable changes in activity. In this project we will also examine the consequences of making specific changes, using transcription reactions reconstituted with purified components and plant genetic complementation experiments. The outcome will be a better understanding of what role each component of PEP has, how it performs it, and why these processes are essential to chloroplast development and photosynthesis.This project is expected to deepen our fundamental understanding of the biochemical basis of transcription. Decades of detailed study have been performed on the proteins that perform transcription in the eukaryotic nucleus and bacteria. This has shown that collating information about diverse proteins is essential to inferring general principles of how gene expression is regulated. Understanding the unique set of proteins that act on chloroplast genes therefore represents an exciting opportunity to advance this. Transcription regulation is a key component to human health and disease, and this research consequently has diverse potential uses. Photosynthesis has a central role in producing the oxygen and energy that sustains much of life on earth. Detailed structural and biochemical studies on the photosynthetic proteins have revealed in detail how they harness solar energy, and this has provided a valuable foundation for crop improvement and development of diverse biotechnologies. By contrast, equivalent mechanistic studies of the gene expression processes that underpin production of the photosynthetic proteins are largely lacking. This project will answer a complementary set of questions: what determines the timing and level of photosynthetic protein production, and how could we modify this to develop more robust crops and new biotechnological applications?