Poster abstracts

Poster number 139 submitted by Michael Wolfe

In vivo characterization of multimer-disrupting variants of the Human Thg1 enzyme

Michael Wolfe (Biochemistry and Chemistry Department, Ohio State University), Bill Eberley (Biochemistry and Chemistry Department, Ohio State University), Jane Jackman (Biochemistry and Chemistry Department, 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 similarities between canonical DNA/RNA polymerases and eukaryotic Thg1 suggest unexpected connections between these enzyme activities. Recent structure and transient kinetic analysis of human Thg1 for incorporation of the guanine residue to the -1 position (G-1) of tRNAHis reveals several mechanistic features of 3’-5’ addition. 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, similar in overall molecular weight to the mass of a single Thg1 monomer, andThg1 has been observed to exist as a tetramer, 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 that are 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.

Keywords: Thg1, tRNAHis