Browsing by Creator "Funk, Holly M."
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Item Identification of the enzyme required for the acp3U modification in plastidic tRNA(Northern Kentucky University) Spigelmyer, Sarah; Funk, Holly M."Posttranscriptional tRNA modifications are essential for protein translation and proper cell growth. Defects in human tRNA modifications are associated with diseases such as cancer, type two diabetes, neurological disorders, and mitochondrial-linked disorders. The 3-amino-3 propylcarboxyuridine (acp3U) modification on tRNA is found in bacteria, plants, and animals, but not in yeast. DTWD proteins were recently identified as the enzymes responsible for acp3U modifications on eukaryotic tRNA. DTWD2 was shown to be responsible for the acp3U modification at residue 20a in human cells. We determined that DTWD2 forms acp3U20a in D. melanogaster cells. In A. thaliana, there are three homologs of human DTWD2 which are DTWD2A (AT2G41750), DTWD2B (AT5G54880), and TapT-like (AT1G03687). Our lab was able to identify that the aspartate and tryptophan residues of the DTWD2A protein in A. thaliana are required for modification activity and we were able to determine that it forms acp3U at position 20b on the tRNA. This was determined by expressing A. thaliana tRNA and A. thaliana DTWD2 genes in trm1Δ met22Δ yeast cells and testing by primer extension with fluorescent oligonucleotides. It is the current aim of this work to utilize the methods used to identify DTWD2A as the enzyme responsible for acp3U at position 20b to identify the substrates of the DTWD2B and Tap-T like proteins. From this research we hope to better understand gene expression in multiple model multicellular eukaryotes."Item Identification of Trm7 residues required for Trm734 binding for tRNA methyltransferase activity in yeast(Northern Kentucky University) Le, Thao Linh (Rosie); Fraley, Morgan E.; Le, Linh; Mullins, Alex K.; Funk, Holly M.; Bekele, Bereket T.; Guy, Adrian R.; Guy, Michael P."Post-transcriptional modifications are made on tRNA, an important molecule for the synthesis of proteins, that allow for functional proteins and cell growth. The enzymes that are involved in the modification process are still being identified in higher eukaryotes, including humans. In yeast, it has been shown that Trm7 forms a complex with Trm732 and Trm734 to modify tRNA at positions 32 and 34, respectively. In humans, the lack of Trm7 causes intellectual disability. The crystal structure of the Trm7- Trm734 complex has been solved, however, the Trm7 residues that are important for the interaction for these proteins are still being investigated. We have identified Trm7 Y138 as important for binding to Trm734 using site-directed mutagenesis and a spot test assay. We are now testing another 10 residues that are predicted to be important for binding based on the crystal structure. Understanding this interaction can be used to identify Trm7 residues important for the Trm7- Trm732 interaction, whose structure has yet to be solved."