The Recognition of tRNA
Our long standing interest in the interaction of tRNA with various components of the translation apparatus is currently focused in two areas.
(1) We have developed a new assay for aminoacyl tRNA synthetases that permits us to follow the attachment of the amino acid onto tRNA under pre-study state (enzyme excess) conditions. This will allow us to examine the reaction in a way that more closely reflects the physiological situation and evaluate the activity of modified tRNAs in a more meaningful fashion.
(2) We have prepared a series of derivatives of yeast tRNA Phe which contain single deoxynucleotides and evaluated their ability to bind elongation factor Tu. Of the eight 2' hydroxyl groups predicted by the crystal structure to make contact with the protein, only four have a thermodynamic effect when changed to a deoxynucleotide. We are currently evaluating why the other four do not show an effect. In addition, there is one 2' hydroxyl that affects protein binding by stabilizing the structure of the tRNA in its bound form. This same set of 2' hydroxyl modified tRNAs have revealed that EF-1a, the eukaryotic homologue of EF-Tu, interacts with tRNA in a similar but distinct manner. In the future, we will examine the activity of these modified tRNAs with other enzymes that interact with tRNA, including the ribosome.