Multi-Synthetase Complex Formation in Methanothermobacter thermautotrophicus

First place at Denman Undergraduate Research Forum, Biological Sciences Category, 2006Award winner at Biological Sciences Colloquium, 2006Aminoacyl-tRNA synthetases (aaRSs) are the enzymes responsible for attaching free amino acids to cognate tRNA. This reaction is performed in two steps. First the specific amino acid is activated by reacting with ATP, forming the aminoacyl-AMP intermediate. Next, the aminoacyl-AMP is transferred from the aaRS to the tRNA forming aminoacyl-tRNA. Now the aaRS is free to catalyze other attachments. In eukaryotic organisms, aaRSs are commonly found in multi-enzyme complexes within the cell. Although the function of these complexes remains unknown, it has been suggested that these interactions may contribute to multiple functions including augmenting catalytic capabilities of the synthetases, RNA processing, and apoptosis (4). A complex consisting of prolyl- and leucyl-tRNA synthetases (ProRS, LeuRS) with enhanced catalytic abilities of ProRS was recently discovered in the archaeal species Methanothermobacter thermautotrophicus (6). This study is investigating the hypothesis that leucyl- and lysyl-tRNA synthetases (LeuRS, LysRS) form a complex in M. thermautotrophicus. Also, because of the known association between LeuRS and ProRS in M. thermautotrophicus, this study seeks to determine whether ProRS and LysRS, which both bind LeuRS, possibly interact with each other as well (4). Having an understanding of the components in these complexes may lead to further insight as to the role they play in the cell. The interaction between these enzymes was investigated using affinity co-purification experiments and aminoacylation assays. Co-purification experiments indicate that LysRS:LeuRS and LysRS:ProRS co-purify together. Aminoacylation assays are used to determine the steady-state aminoacylation kinetics, an indicator of the efficiency of the enzyme both alone, and in complexes. The results from these assays indicate that LysRS activity is enhanced by LeuRS and ProRS. Current work is focused on optimizing fluorescent anisotropy experiments to study complex formation between the two enzymes. Tests are underway to determine if changes in anisotropy can be detected when LeuRS is incubated with LysRS. This project is significant to understanding the role of the aminoacyl-tRNA synthetases in the cell and the interactions they are involved in that help to maintain the fidelity of the genetic code.Dean's Undergraduate Research Fund2005 Mayers Summer InternshipCollege of the Arts and Sciences 2005 Undergraduate Research Scholarshi