Structure and function of the chloroplast transcription machinery

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没有很好的理解。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。尽管它们的重要性，叶绿体基因表达机制的功能还没有被详细描述。该项目的重点是叶绿体中基因表达的第一阶段，其中基因被转录以产生编码光合蛋白的信使RNA（mRNA）。叶绿体转录的调控是植物发育的一个关键阶段：叶绿体对光的反应成熟。这一过程在植物变绿色的过程中可以观察到，当有太阳能可供光合作用蛋白质收集时，光合作用蛋白质可以选择性地产生。然而，叶绿体转录是如何被激活的还不清楚。为了进一步了解叶绿体转录及其激活机制，我们将对执行这一过程的酶：质体编码的聚合酶（PEP）进行分析。PEP是一个至少有16个蛋白质亚基的大分子组装体。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，稳定结合到核心的12种或更多蛋白质在核基因组中编码并输入叶绿体。因此，我们期望这些蛋白质，被称为PAP（PEP相关蛋白），编排的关键调控过程独特的叶绿体。在这个项目中，我们将可视化的叶绿体转录机制的分子结构，使用低温电子显微镜（cryo-EM）。PEP的原子模型将阐明每个亚基如何调节转录以响应叶绿体的需要。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。我们将研究的后果，使特定的变化，使用转录反应重组纯化成分和植物遗传互补实验。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. Yet how plants produce their photosynthetic proteins is not well understood. 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. This project focuses on the first stage of gene expression in the chloroplast, in which genes are transcribed to produce messenger RNAs (mRNAs) that encode photosynthetic proteins. Regulation of chloroplast transcription underpins a key stage of plant development: the maturation of chloroplasts in response to light. This process is observable in the plant turning green and allows photosynthetic proteins to be selectively produced when solar energy is available for them to collect. Yet how chloroplast transcription is activated is not well understood.To advance our understanding of chloroplast transcription and the mechanism of its activation, we will characterise the enzyme that performs this process: the plastid-encoded polymerase (PEP). PEP is a large molecular assembly with at least 16 protein subunits. 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 and imported to the chloroplast. We therefore expect that these proteins, known as PAPs (PEP-associated proteins), orchestrate key regulatory processes unique to the chloroplast.In this project we will visualise the molecular architecture of the chloroplast transcription machinery using cryogenic electron microscopy (cryo-EM). Atomic models of PEP will shed light on how each subunit regulates transcription in response to the needs of the chloroplast. 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. We will 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?