Poster abstracts

Poster number 60 submitted by Bhalchandra Rao

Biochemical and structural characterization of B. thuringiensis tRNAHis guanylyltransferase (BtThg1)

Bhalchandra S. Rao (Biochemistry, The Ohio State University), Maria Abad (Biochemistry, The Ohio State University), Charles E. Bell (Molecular and Cellular Biochemistry, The Ohio State University), Jane E. Jackman (Biochemistry, The Ohio State University)

Abstract:
Addition of a single G_-1 to the 5' end of tRNAHis, in the 3'-5' direction, is an essential and universally conserved modification. This activity is required for recognition of tRNA^His by HisRS and subsequent aminoacylation. The enzyme that catalyzes this reaction, tRNA^His guanylyltransferase (Thg1), was first identified in S.cerevisiae.
G_-1 addition in prokarya differs from eukarya as it is Thg1 independent because G_-1 is genomically encoded. Nevertheless, protein sequences homologous to yeast Thg1 are found in several bacterial organisms. Since a role for Thg1 in tRNA^His maturation is not predicted in bacteria, function of these homologs is unclear. To address this, we have cloned B.thuringiensis Thg1 (BtThg1) to compare its biochemical activities to those of yeast Thg1. We determined that BtThg1 can catalyze G_-1 addition but exhibits differences from yeast Thg1. To quantify these differences, we measured steady state kinetic parameters with ppptRNA^His and ppptRNA^Phe, which have already been characterized as yeast Thg1 substrates. BtThg1 exhibits K_M for tRNA^His similar to K_M for tRNA^Phe indicating a lack of selective recognition of tRNA^His over other tRNA species, consistent with lack of involvement of Thg1 in tRNA^His processing in bacteria. These data imply a possibility of a hitherto unknown function associated with Thg1.
A second major goal of our research is structural characterization of Thg1 for which no structural information exists. This investigation will provide key insights into mechanisms of Thg1 substrate recognition, binding and catalysis at a molecular level. BtThg1 has been promising in this aspect. We have been able to crystallize the protein and efforts are underway to generate diffraction data and solve its crystal structure. We expect that structural characterization will reveal a multimeric Thg1 enzyme, since AUC sedimentation velocity analysis indicates that BtThg1 exists as a tetramer in solution. This is the first physical demonstration of a multimeric state of Thg1 and further investigation into the involvement of quaternary structure in activity is ongoing.

Keywords: Thg1, tRNAHis