Poster abstracts

Poster number 53 submitted by Michael Wolfe

In vivo characterization of multimer-disrupting variants of human Thg1

Michael Wolfe (Ohio State University), Bill Eberley (Ohio State University), Jane Jackman (Ohio State University)

Abstract:
The tRNAHisguanylyltransferase (Thg1) is part of a family of enzymes found in all three domains of life, members of which catalyze an unprecedented 3’—5’ nucleotide addition reaction. Structural and transient kinetic analysis of human Thg1 reveals several mechanistic features of 3’-5’ addition, including a striking similarity between Thg1 and canonical DNA/RNA polymerases. However, many mechanistic questions, particularly related to the ability of Thg1 to bind to its tRNA substrates, remain unsolved. Since tRNA is a relatively large molecule, we hypothesize that the multimeric form of the enzyme is an important feature allowing recognition of the large tRNA substrate. For this reason, we chose to investigate several highly conserved residues predicted to be involved in Thg1 multimerization. Previously, variants of human Thg1 believed to disrupt the dimer-dimer interface of the enzyme based on their positions in the crystal structure have been characterized in vitro. The purpose of this project was to test the function of these particular variants in vivo in yeast. This was accomplished using a yeast complementation assay to test whether the variant was able to support growth in the absence of the wild type THG1 gene. The in vivo results were found to largely mirror the previous in vitro characterization, supporting the predicted role of these particular residues in holding the dimer-dimer interface together. Future research will include the in vitro characterization of new residues of interest that will hopefully shed light on the functional residues in the enzyme.

Keywords: Thg1