RNA repair pathways rely on RNA ligases to maintain or manipulate RNA structure in response to purposeful breakage events inflicted during physiological RNA processing (e.g. tRNA splicing; mRNA editing) and under conditions of cellular stress (e.g., virus infection, unfolded protein response). The goal of this project is to illuminate the mechanisms and structures of enzymes that repair broken RNA ends. Our proposed studies embrace two RNA repair systems that exploit ATP-dependent RNA ligases. (1) Fungal and plant tRNA ligases are essential for tRNA splicing and for mRNA splicing during in the unfolded protein response. A multifunctional tRNA ligase enzyme heals and seals the ends of the tRNA exons via the sequential action of 2',3'-cyclic phosphodiesterase, 5' polynucleotide kinase, and ATP-dependent RNA ligase catalytic domains. The defining feature of fungal/plant tRNA ligases is their requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. The structural basis for this requirement is uncharted. Because mammals rely on a completely different biochemical pathway of tRNA exon splicing, and mammalian proteomes have no homologs of the fungal ligase domain, we regard fungal tRNA ligases as promising targets for antifungal drug discovery. To advance that end, we propose to structurally characterize the catalytic domains of tRNA ligases from several fungal species, including the human pathogens Aspergillus fumigatus, Candida albicans, and Coccidioides immitis. (2) Naegleria gruberi RNA ligase (NgrRnl), is the founder of a new family of RNA nick-sealing enzymes ? found in bacteria, viruses, fungi, and protozoa ? in which a nucleotidyltransferase module (common to all ATP- dependent ligases) is fused to a signature N-terminal module not found in any other RNA ligase clade. Our structures of the NgrRnlATP(Mn2+)2 Michaelis complex and the NgrRnl-(lysyl-N)?AMPMn2+ covalent intermediate suggest a two-metal mechanism of ligase adenylylation. We will use these structures to guide functional studies of the ligase reaction mechanism. To understand the basis for nick recognition and phosphodiester synthesis, we propose to capture structures of NgrRnl in complexes with nicked duplex substrates.
tRNA ligases are essential agents of informational and stress response pathways involving the repair of tRNA breaks (during tRNA splicing) and mRNA breaks (during the unfolded protein response). The biochemical mechanism of fungal tRNA ligase (Trl1) is completely different from that of human tRNA ligase (RtcB), a situation that recommends Trl1 as target for anti-fungal drug discovery. To advance that end, we propose to structurally characterize tRNA ligases from several fungal species, including the human pathogens Aspergillus fumigatus, Candida albicans, and Coccidioides immitis. Aspergillosis and Candidiasis are serious and frequently fatal infections of immunocompromised hosts, often associated with cancer (hematological malignancies) and cancer treatment (hematological stem cell transplantation). C. immitis is the causative agent of San Joaquin Valley Fever, a major public health problem in the US desert Southwest region.
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