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?

植物生长是由光合作用驱动的。然而，植物如何产生光合蛋白尚不清楚。叶绿体中包含一个基因组，该基因组编码了关键的光合蛋白和表达它们的独特分子机械。尽管它们的重要性，但叶绿体基因表达机制的功能尚未详细表征。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）。叶绿体转录的调节是植物发育的关键阶段：叶绿体的成熟响应光。该过程在植物变绿的植物中可以观察到，并在太阳能可供收集时，可以选择性地生产光合蛋白。然而，尚不清楚叶绿体转录的激活方式。为了促进我们对叶绿体转录及其激活的机制的理解，我们将表征执行此过程的酶：质体编码的聚合酶（PEP）。 PEP是一种具有至少16个蛋白质亚基的大分子组件。在转录酶中，PEP是显着的，因为它包含两个进化起源的亚基。核心类似于细菌酶，并用蓝细菌祖先的叶绿体基因组遗传。相比之下，稳定结合到核心的十二个或更多的蛋白质被编码在核基因组中并进口到叶绿体。因此，我们期望这些蛋白质（称为PAPS（PEP相关蛋白））编排叶绿体特有的关键调节过程。在该项目中，我们将使用低温电子显微镜（Cryo-Em）可视化叶绿体转录机械的分子结构。 PEP的原子模型将阐明每个亚基如何根据叶绿体的需求调节转录。现代冷冻EM提供的细节水平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设计精确的修改。我们将使用与纯化的成分和植物遗传互补实验重构的转录反应来检查进行特定变化的后果。结果将更好地理解PEP的每个组成部分的作用，其执行方式以及这些过程为何对叶绿体的发展和光合作用至关重要。期望这项项目可以加深我们对转录生化基础的基本理解。数十年的详细研究已经对在真核核和细菌中进行转录的蛋白质进行了进行。这表明，关于多种蛋白质的整理信息对于推断基因表达的调节方式的一般原理至关重要。因此，了解对叶绿体基因作用的独特蛋白质集代表了促进它的激动人心的机会。转录调节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。光合作用在生产地球上大部分生命的氧气和能量方面具有核心作用。有关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，这为作物改善和发展的多种生物技术提供了宝贵的基础。相比之下，在很大程度上缺乏基于光合蛋白产生的基因表达过程的同等机理研究。该项目将回答一系列互补的问题：什么决定了光合蛋白质生产的时机和水平，我们如何对此进行修改，以开发更多可靠的农作物和新的生物技术应用？