Poster abstracts

Poster number 73 submitted by Brian Smith

Kinetic characterization of the adenylation of yeast tRNAHis catalyzed by tRNAHis guanylyltransferase (Thg1)

Brian Smith (Biochemistry, The Ohio State University), Jane Jackman

Abstract:
tRNA^His guanylyltransferase (Thg1) is an essential enzyme in yeast that catalyzes incorporation of a single guanosine residue at the -1 position (G_-1) of tRNA^His. G_-1 serves as a necessary recognition element for histidyl-tRNA synthetase and is conserved among all tRNA^His species. Thg1 is the only known enzyme that adds nucleotides in the 3'-5' direction and shares no identifiable sequence homology to any other known enzyme, thus its molecular mechanism is unknown.
The first step in G_-1 addition involves activation of the 5' end of monophosphorylated tRNA^His by formation of an adenylated intermediate. In subsequent steps the 3'-OH of GTP attacks the intermediate yielding AMP and resulting in the addition of a single GTP to the 5' end of the tRNA. In the final step Thg1 removes pyrophosphate from the G_-1 residue yielding mature G_-1-containing tRNA^His. A complete understanding of this complex reaction mechanism requires isolation and characterization of each of these catalytic steps individually. Previous studies suggest that adenylation is a major specificity determining step since Thg1 is unable to adenylate tRNA substrates lacking the tRNA^His anticodon (GUG). Therefore, we have first focused on the kinetic characterization of the adenylation reaction. We used transient kinetic techniques (single turnover assays) to explicitly measure the first-order rate constant of adenylation (k_aden) of tRNA^His for wild type yeast Thg1. We measured a k_aden of 0.25 s^-1, which is significantly faster than the previously measured k_cat (0.012 s^-1), suggesting that adenylation is not the rate determining step for G_-1 addition under these conditions. Our results suggest a rapid equilibrium mechanism for adenylation of tRNA^His (k_-1 >> k_aden), with K_{D app}~ 0.88 µM. To identify catalytic residues that participate in adenylation, we are currently using site directed mutagenesis to identify Thg1 variants that exhibit compromised adenylation activity however still retain that ability to carry out guanylyltransfer.

Keywords: tRNAHis, Thg1