Structure of Malaria Parasite RNA polymerase

疟疾寄生虫 RNA 聚合酶的结构

• 批准号: 10433276
• 负责人: Manuel Llinas
• 金额: $ 23.73万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-21 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10433276
• 关键词: AIDS/HIV problem; Accounting; Affinity; Affinity Chromatography; Antibiotics; Antimalarials; Asparagine; Aspartate; Bacteria; Bacteriophages; Binding; Biochemical; Biological Assay; Biological Process; COVID-19 treatment; CRISPR/Cas technology; Cell Nucleus; Cells; Cessation of life; Child; Clostridium difficile; Code; Cryoelectron Microscopy; DNA; DNA-Directed RNA Polymerase; Development; Diarrhea; Disease; Drug Design; Drug Screening; Drug Targeting; Enzymes; Eukaryota; Evolution; Future; Gene Expression; Genetic Transcription; Genome; Genomic DNA; Goals; Human; In Vitro; Infection; Intervention; Joints; Laboratories; Life; Maintenance; Malaria; Messenger RNA; Methods; Mitochondrial RNA; Nuclear; Nuclear Extract; Nuclear RNA; Organelles; Parasites; Persons; Pharmaceutical Preparations; Plasmodium; Plasmodium falciparum; Play; Pregnant Women; Production; RNA; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; RNA chemical synthesis; Research; Research Project Grants; Resistance development; Resolution; Rifampin; Role; Small Nuclear RNA; Specimen; Stretching; Structure; System; Techniques; Testing; Tuberculosis; Untranslated RNA; Vaccines; Virus; Virus Replication; Work; Zidovudine; affinity labeling; antiviral drug development; arginyllysine; base; design; drug action; drug candidate; enzyme structure; genetic manipulation; genome editing; in silico; in vitro activity; inhibitor; insight; lead candidate; new therapeutic target; novel; particle; pathogen; protein structure; public health relevance; remdesivir; scaffold; screening; stable cell line; three dimensional structure

Project Summary Malaria, the most wide-scale protozoan-induced infection of humans, is caused by parasites from the genus Plasmodium, with Plasmodium falciparum being responsible for the most severe form of disease in humans. Although there are several drugs that are currently used to treat malaria, malaria parasites have rapidly developed resistance to all currently available frontline drugs. This demonstrates that there is an urgent and critical need to identify novel drug targets for the development of new and effective anti-malarial drugs. Transcription of DNA into RNA, the first step of gene expression, is carried out by RNA polymerase (RNAP) in all life forms and viruses. Due to its essential role in life maintenance and virus replication, RNAP is a proven drug target for antibiotic and antiviral developments. The biochemical characterization of purified RNAP in vitro along with structural studies have played essential roles to define our understanding of the structure and function of RNAP. Purified RNAP has also facilitated large-scale drug screening and the characterization of drug candidate lead compounds, and structural studies of RNAPs have revealed the mechanism of drug action as well as accelerated drug design by in silico screening. RNA synthesis in Plasmodium parasites occurs in three organelles (nucleus, mitochondrion and non- photosynthetic apicoplast) and is carried out by five RNAPs including three nuclear RNAPs (RNAP I, RNAP II and RNAP III), bacteriophage-type mitochondrial RNAP and bacterial-type apicoplast RNAP. The ultimate goal of this research will be to isolate all endogenous nuclear RNAPs from P. falciparum cells and determine the 3D structures of these enzymes by single-particle cryo-electron microscopy (cryo-EM). These high-resolution structures will elucidate the mechanism of transcription in Plasmodium and create valuable platforms for the design of new antimalarial drugs targeting P. falciparum RNAPs. Finally, these structures will also provide new insight into the evolution of RNAPs during the course of parasitic adaptation in eukaryotes. In this proposal, we seek to establish methods to investigate the structure and function of P. falciparum RNAP II, which is responsible for mRNAs, snRNAs and regulatory RNAs transcription. Aim 1 will use CRISPR/Cas9 genome editing to generate a stable cell line of P. falciparum expressing an affinity-labeled largest subunit (Rpb1) of RNAP II. Aim 2 will establish a purification method of RNAP II from P. falciparum nuclear extract using a combination of chromatographic techniques that will be tracked by an in vitro transcription assay to test RNAP II activity. In Aim 3, we will determine the atomic-resolution 3D structure of RNAP II by cryo-EM. The proposed work will pave the way for investigating the structure and function of all three nuclear RNAPs from Plasmodium.

项目摘要 疟疾是最广泛的原生动物引起的人类感染，是由寄生虫引起的。 疟原虫，恶性疟原虫是人类最严重的疾病。 尽管目前有几种药物用于治疗疟疾，但疟疾寄生虫迅速地 对所有现有的一线药物都产生了抗药性。这表明，有一个紧迫的和 迫切需要确定新的药物靶点，以开发新的和有效的抗疟疾药物。 DNA转录成RNA是基因表达的第一步，通过RNA聚合酶（RNAP）在细胞中进行。 所有的生命形式和病毒由于其在生命维持和病毒复制中的重要作用，RNAP是一种经过验证的 抗生素和抗病毒药物的发展目标。纯化RNAP的体外生化特性 沿着结构研究，它在定义我们对结构和功能的理解方面发挥了重要作用 关于RNAP纯化的RNAP还促进了大规模药物筛选和药物表征 候选先导化合物，RNAP的结构研究揭示了药物作用机制， 以及通过计算机筛选加速药物设计。 疟原虫的RNA合成发生在三种细胞器（细胞核、胞核和非胞核）中。 光合顶质体），并进行了五个RNAP，包括三个核RNAP（RNAP I，RNAP II 和RNAP III）、噬菌体型线粒体RNAP和细菌型顶质体RNAP。最终目标 本研究的目的是从恶性疟原虫细胞中分离所有内源性核RNAP，并确定3D 这些酶的结构通过单粒子冷冻电子显微镜（cryo-EM）。这些高分辨率 这些结构将阐明疟原虫中的转录机制，并为疟原虫的研究创造有价值的平台。 设计针对恶性疟原虫RNAP的新型抗疟药物。最后，这些结构还将提供新的 深入了解RNAP在真核生物寄生适应过程中的进化。 在本研究中，我们试图建立研究恶性疟原虫RNAP结构和功能的方法， II，负责mRNA、snRNA和调节RNA的转录。Aim 1将使用CRISPR/Cas9 基因组编辑以产生表达亲和标记的最大亚基（Rpb 1）的恶性疟原虫的稳定细胞系 关于RNAP II目的2建立恶性疟原虫核提取物RNAP Ⅱ的纯化方法， 将通过体外转录测定跟踪以测试RNAP II的色谱技术的组合 活动在目标3中，我们将通过cryo-EM确定RNAP II的原子分辨率3D结构。拟议 这项工作将为研究疟原虫所有三种核RNAP的结构和功能铺平道路。