Structure of Malaria Parasite RNA polymerase

疟疾寄生虫 RNA 聚合酶的结构

• 批准号: 10552645
• 负责人: Manuel Llinas
• 金额: $ 19.77万
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10552645
• 关键词: Acceleration; Accounting; Affinity; Affinity Chromatography; Antibiotics; Antimalarials; Arginine; Asparagine; Aspartate; Bacteria; Bacteriophages; Binding; Biochemical; Biological Assay; Biological Process; COVID-19 treatment; CRISPR/Cas technology; Cell Nucleus; Cells; Cessation of life; Child; Chromatography; 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; Genomic DNA; Goals; HIV/AIDS; Human; In Vitro; Infection; Intervention; Joints; Laboratories; Life; Lysine; Maintenance; Malaria; Messenger RNA; Methods; Mitochondrial RNA; Nuclear; Nuclear Extract; Nuclear RNA; Organelles; Parasites; Persons; Pharmaceutical Preparations; Plasmodium; Plasmodium falciparum; Plasmodium falciparum genome; Play; Pregnant Women; Production; RNA; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; RNA chemical synthesis; RNA purification; 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; 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 疟疾是由原生动物引起的最大范围的人类感染，由该属的寄生虫引起。 恶性疟原虫是人类最严重的疾病的罪魁祸首。 尽管目前有几种药物用于治疗疟疾，但疟疾寄生虫迅速 对目前所有可用的一线药物产生抗药性。这表明有一个紧迫的和 迫切需要确定新的药物目标，以开发新的有效的抗疟疾药物。 DNA转录成RNA是基因表达的第一步，由RNA聚合酶(RNAP)在 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开发的靶点。纯化的RNAP体外生化特性研究 与结构研究一起发挥了至关重要的作用，定义了我们对结构和功能的理解 RNAP。纯化的RNAP还促进了大规模药物筛选和药物表征 候选先导化合物和RNAP的结构研究揭示了药物作用的机制如下 以及通过计算机筛查加速药物设计。 疟原虫的RNA合成存在于三个细胞器(细胞核、线粒体和非核细胞)中。 光合质外体)，由5个RNAP执行，其中包括3个核RNAP(RNAP I、RNAP II 和RNAP III)、噬菌体型线粒体RNAP和细菌型质外体RNAP。终极目标 这项研究的重点将是从恶性疟原虫细胞中分离所有内源性RNAP，并确定3D 用单粒子低温电子显微镜(Cryo-EM)观察这些酶的结构。这些高分辨率 结构将阐明疟原虫的转录机制，并为 针对恶性疟原虫RNAPs的新型抗疟药物的设计。最后，这些结构还将提供新的 真核生物寄生适应过程中RNAPs的进化。 在这项建议中，我们试图建立方法来研究恶性疟原虫RNAP的结构和功能 II，负责mRNAs、SnRNAs和调控RNAs的转录。AIM 1将使用CRISPR/CAS9 基因组编辑以获得稳定表达亲和标记最大亚基(Rpb1)的恶性疟原虫细胞系 目的建立一种从恶性疟原虫核提取液中分离纯化RNAP II的方法。 将通过体外转录试验跟踪检测RNAP II的层析技术组合 活动。在目标3中，我们将用冷冻电子显微镜测定RNAP II的原子分辨三维结构。建议数 这项工作将为研究来自疟原虫的所有三个核RNAP的结构和功能铺平道路。