Poster abstracts
Poster number 49 submitted by Tracy Roach
Recognition of RNA substrates by Thg1 family proteins
Tracy M. Roach (Ohio State Biochemistry Program)
Abstract:
tRNAHis guanylytransferase (Thg1) is responsible for adding a G-1 to tRNAHis across from an A73 discriminator nucleotide. This step, and the addition of CCA to the 3'-end of tRNAHis, is required for histidyl tRNA synthetase to recognize and aminoacylate tRNAHis. Because both Thg1 and CCA adding enzyme localize to the cytosol of S. cerevisiae, these two essential enzymes would likely compete for the end-matured tRNAHis substrate, and therefore it is of interest to understand whether there is a preferred pathway in terms of which enzyme is acting on tRNAHis first. Identification of any obligate processing order will define the biologically relevant substrates for each enzyme. To evaluate these possibilities, kinetic studies were used to determine rates of nucleotide incorporation to the 5'-end of tRNA substrates containing and lacking the CCA 3'-end sequence. Here we report that the in vitro studies support a preferred pathway in which CCA addition precedes 5'-end maturation by Thg1 in yeast. To further understand substrate recognition by eukaryotic Thg1 enzymes, an unusual eukaryotic Thg1 enzyme is being explored that appears capable of forming an alternative quaternary structure to the homotetrameric enzyme that has been observed in all other species to date. Efforts to define the catalytically active composition of the Thg1 enzyme in these species will be used as the basis for individual manipulation of one domain of an active Thg1 protein complex at a time to understand the contributions of different subunits to catalysis.
Keywords: Thg1, tRNA processing, enzymology