Defining the molecular basis of chloroplast transcription of photosynthetic genes

定义光合基因叶绿体转录的分子基础

• 批准号: BB/Y003802/1
• 负责人: Michael Webster
• 金额: $ 83.95万
• 依托单位: John Innes Centre
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY003802%2F1
• 关键词: Defining; molecular; basis; chloroplast; transcription

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?

植物的生长是由光合作用驱动的。然而，人们尚不清楚植物如何产生光合蛋白质。叶绿体包含编码关键光合蛋白质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很重要，但其功能如何尚未得到详细描述。叶绿体基因产生光合蛋白的第一步是转录产生信使 RNA (mRNA)。这个过程是由称为质体编码聚合酶（PEP）的大量蛋白质组装完成的。由于 PEP 转录活性的激活，植物响应光而变绿。此外，干旱、高温和病原体攻击等植物胁迫会影响 PEP 活性，从而使编码光合蛋白的特定基因打开或关闭。尽管 PEP 在植物发育和适应中发挥着核心作用，但人们对 PEP 如何转录叶绿体基因却知之甚少。 PEP 由 19 种不同的蛋白质亚基组成，每个亚基都发挥着重要作用。 PEP 在转录酶中非常引人注目，因为它包含两个进化起源的亚基。其核心类似于细菌酶，并与蓝藻祖先的叶绿体基因组一起遗传。相比之下，稳定结合到核心的十二种或更多蛋白质是在核基因组中编码的。因此，我们期望这些被称为 PAP（PEP 相关蛋白）的蛋白质能够协调叶绿体特有的关键调控过程。为了更好地了解植物如何产生光合蛋白质，我们的目标是在 PEP 转录基因时将其可视化。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。现代冷冻电镜提供的详细程度对于开发新假设非常有价值，因为可以通过可预测的活动变化来设计精确的修改。在这个项目中，我们还将使用纯化成分重构的转录反应和植物遗传互补实验来研究进行特定改变的后果。结果将是更好地理解 PEP 的每个组成部分的作用、它如何发挥作用，以及为什么这些过程对叶绿体发育和光合作用至关重要。该项目预计将加深我们对转录生化基础的基本理解。对在真核细胞核和细菌中进行转录的蛋白质进行了数十年的详细研究。这表明整理有关不同蛋白质的信息对于推断基因表达如何调节的一般原理至关重要。因此，了解作用于叶绿体基因的一组独特蛋白质为推进这一目标提供了令人兴奋的机会。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，对支持光合蛋白产生的基因表达过程的等效机制研究却在很大程度上缺乏。该项目将回答一系列补充问题：什么决定了光合蛋白质产生的时间和水平，以及我们如何修改它以开发更强大的作物和新的生物技术应用？