Structure and function of the chloroplast transcription machinery

叶绿体转录机制的结构和功能

• 批准号: MR/X033481/1
• 负责人: Michael Webster
• 金额: $ 195.12万
• 依托单位: John Innes Centre
• 依托单位国家: 英国
• 项目类别: Fellowship
• 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=MR%2FX033481%2F1
• 关键词: Structure; function; chloroplast; transcription; machinery

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?

植物的生长是由光合作用驱动的。然而植物如何产生光合蛋白质尚不清楚。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，但其如何发挥作用尚未得到详细描述。该项目重点研究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)。叶绿体转录的调节是植物发育的一个关键阶段的基础：叶绿体响应光的成熟。这个过程可以在植物变绿的过程中观察到，并且当植物可以收集太阳能时，可以选择性地产生光合蛋白质。然而，叶绿体转录是如何激活的尚不清楚。为了加深我们对叶绿体转录及其激活机制的理解，我们将描述执行此过程的酶：质体编码聚合酶（PEP）。 PEP 是一个至少包含 16 个蛋白质亚基的大分子组装体。 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在核基因组中编码并输入叶绿体。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。在这个项目中，我们将使用低温电子显微镜 (cryo-EM) 可视化叶绿体转录机制的分子结构。 PEP 的原子模型将揭示每个亚基如何调节转录以响应叶绿体的需求。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。我们将使用纯化成分重构的转录反应和植物遗传互补实验来检查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？