Mechanisms of DNA and RNA Transactions

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PROJECT SUMMARY: The goals of the research proposed for the MIRA renewal are: (i) to understand the mechanisms and structures of enzymes that perform nucleic acid synthesis, modification, and repair; and (ii) to elucidate factors that regulate these events. The project integrates diverse experimental approaches (microbiology, biochemistry, structural biology, genetics) and applies them to model systems ranging from viruses to bacteria to fungi. The principal themes are: (1) The structures, mechanisms, and distinctive specificities of fungal tRNA splicing enzymes Trl1 (tRNA ligase) and Tpt1 (tRNA 2'-phosphotransferase) – as paradigms of an RNA repair system essential for normal cell physiology and as promising targets for anti-fungal drug discovery. We will determine structures of Trl1 and Tpt1 in complexes with nucleic acid and nucleotide substrates and cofactors, and endeavor to capture structural snapshots of intermediates and transition-states along the reaction pathways. (2) The structural basis for RNA recognition and strand joining by ATP-dependent 5'-PO4/3'-OH RNA ligase T4 Rnl1. Rnl1 is a tRNA repair enzyme that the T4 bacteriophage uses to evade a tRNA-damaging host response to virus infection. (3) The unique catalytic mechanism, end-specificity, and regulation of GTP-dependent 3'-PO4/5'-OH RNA ligase RtcB. The RtcB-family ligases are found in all phylogenetic domains. They are agents of diverse RNA transactions, including tRNA splicing (in metazoa and archaea), RNA repair (in bacteria), nonspliceosomal mRNA splicing (in the metazoan unfolded protein response), and the formation of chimeric RNAs in human cells that can undergo retrotransposition into the human genome. (4) Tandem transcriptional interference as a controlling factor in fission yeast phosphate homeostasis. The three S. pombe PHO regulon genes are repressed in phosphate-replete cells by transcription in cis of 5’- flanking lncRNAs that interferes with the PHO mRNA promoters. The lncRNA-mediated interference that underlies the repression of pho1 has afforded us a sensitive read-out of genetic influences on 3'- processing/termination and a powerful tool for discovery of agents and regulators of this step of the Pol2 transcription cycle. These influences include: (i) the Pol2 CTD code; (ii) numerous components of the 3'- processing/termination machinery; and (iii) metabolite control by inositol pyrophosphate 1,5-IP8, an intracellular signaling molecule. We propose to investigate the fission yeast Asp1 kinase/pyrophosphatase enzyme that determines IP8 dynamics and the cellular proteins and pathways that connect IP8 to gene expression.