Defining the molecular basis of chloroplast transcription of photosynthetic genes

植物的生长是由光合作用驱动的。然而，植物是如何产生光合作用蛋白的还不是很清楚。叶绿体包含一个编码关键光合蛋白的基因组和一个表达这些蛋白的独特分子机制。尽管它们很重要，但叶绿体基因表达机制是如何发挥作用的还没有详细的描述。从叶绿体基因产生光合蛋白的第一阶段是它们的转录产生信使RNA(MRNAs)。这一过程是由一大群蛋白质组装而成的，这种蛋白质被称为叶绿体编码聚合酶(PEP)。植物对光的反应会变绿，因为PEP的转录活性被激活了。此外，干旱、高温和病原体侵袭等植物胁迫会影响PEP的活性，从而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核心作用，但人们对PEP如何转录叶绿体基因知之甚少。PEP由19个不同的蛋白质亚基组成，每个亚基都有重要的作用。在转录酶中，PEP是值得注意的，因为它包含两个进化起源的亚基。核心类似细菌酶，是从蓝藻祖先的叶绿体基因组中遗传过来的。相比之下，稳定结合到核心的12种或更多的蛋白质在核基因组中编码。因此，我们希望这些蛋白质，被称为PEP相关蛋白(PEP相关蛋白)，协调叶绿体特有的关键调控过程。为了更好地了解植物是如何产生光合作用蛋白的，我们的目标是在PEP转录基因时可视化它。为此，我们将使用低温电子显微镜(Cryo-EM)收集PEP分子的图像。通过对这些图像的处理，可以构建原子分辨率的PEP模型。这些有望展示PAPs如何激活叶绿体转录。现代低温EM提供的细节水平对于开发新的假说非常有价值，因为可以根据活动的可预测变化来设计精确的修改。在这个项目中，我们还将使用由纯化成分重组的转录反应和植物遗传互补实验来检查做出特定变化的后果。其结果将是更好地理解PEP的每个组成部分都有什么作用，它是如何发挥作用的，以及为什么这些过程对叶绿体发育和光合作用是必不可少的。这个项目有望加深我们对转录的生化基础的基本理解。几十年来，人们对在真核细胞和细菌中进行转录的蛋白质进行了详细的研究。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是必不可少的。因此，了解作用于叶绿体基因的一组独特的蛋白质是推动这一进程的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，因此这项研究具有不同的潜在用途。光合作用在产生氧气和能量方面起着核心作用，这些氧气和能量维持着地球上的大部分生命。对光合作用蛋白进行了详细的结构和生化研究，详细揭示了它们是如何利用太阳能的，这为作物改良和开发各种生物技术提供了宝贵的基础。相比之下，对支撑光合作用蛋白质生产的基因表达过程的等效机制研究在很大程度上是缺乏的。这个项目将回答一系列补充问题：是什么决定了光合作用蛋白生产的时间和水平，我们如何才能改变这一点，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?