Structure of Malaria Parasite RNA polymerase

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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.