tRNA is part of the molecular machinery that cells depend on to synthesize proteins from mRNA. Post-transcriptional modification of tRNA molecules is an important process which is vital for synthesis of functional proteins and cell growth. Enzymes that are associated with the modification process are still being identified and characterized in higher eukaryotes, including humans. In yeast it has been shown that Trm7 forms a complex with Trm732 to modify tRNAs at position 32 and with Trm734 to modify position 34. In humans, mutations in the TRM7 human homolog (FTSJ1) cause non-syndromic X-linked intellectual disability. So far, the protein-protein binding sites of these proteins have not been discovered. To identify residues involved in protein-protein binding, we generated Trm7 variants by site-directed mutagenesis, and conducted a yeast spot test assay to test function. Our results suggest that Trm7 amino acid residue Y138 is involved in the Trm7-Trm734 interaction. To confirm the involvement of Y138, tRNA modification levels from cells expressing a Trm7-Y138R variant will be analyzed by UPLC and protein-protein interaction will be tested by immunoprecipitation. As an alternative approach our group is developing a high throughput approach using a yeast-two-hybrid assay to test Trm7 variants for Trm732 and Trm734 binding. Understanding how these proteins interact in yeast should facilitate the study of their conserved homologs in humans.

Identification of Trm7 residues required for Trm732 and Trm734 binding for tRNA methyltransferase activity in yeast

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.