RNA structure and function, RNA-protein interactions, chemical mechanisms of biological reactions, protein synthesis, tRNA synthetases

The fidelity of protein synthesis is dependent on accurate substrate recognition by an essential family of enzymes called the tRNA synthetases. There are typically up to twenty different types of tRNA synthetases in the cytoplasm of each cell. Each is responsible for a single cognate amino acid and covalently attaches it to the correct tRNA. The "charged" tRNA then acts as a shuttle to deliver the amino acid to the ribosome where it can be incorporated into the growing polypeptide chain. Because the tRNA synthetases are essential to every cell, their investigation has potential to address basic research questions and also target important long-term medical issues such as antibiotic discovery and development. New tRNA synthetases can also be designed to incorporate novel amino acids into custom-designed proteins for use as tools and/or therapeutics in biotechnology and medical applications. Our research in tRNA synthetases currently focuses in two broad areas:

• Fidelity of Protein Synthesis. Some tRNA synthetases make mistakes and fail to distinguish between closely related amino acids. However, these tRNA synthetases have evolved mechanisms to proofread their errors and edit their mistakes. One of our research goals is to determine mechanistic details and molecular interactions that govern tRNA synthetase aminoacylation and amino acid editing activities. We have made considerable progress in this area using leucyl-tRNA synthetase (LeuRS) as a model. The LeuRS editing active site resides within a discretely folded domain that is called CP1 and is distinct from the aminoacylation active site. Using combinations of biochemical, structural, computational and genetic approaches, we have localized and begun to map the amino acid editing active site and also the aminoacylation active site. Our research has identified molecular determinants that dictate specificity and also influence LeuRS enzymatic activity. Significantly, we have also been able to block the amino acid editing activity, which allows us to activate and stably charge unnatural amino acids to tRNA. These "mischarged" tRNAs can be used to expand the genetic code by introducing novel amino acids into proteins.
  
  
• Novel Activities of tRNA Synthetases. The tRNA synthetases are thought to be one of the most ancient families of proteins. Because of their lengthy evolutionary period, they have often been adopted for other activities in the cell that extend beyond their role in aminoacylation and protein synthesis. A second focus of our research group is to understand the molecular basis of tRNA synthetases in alternate and novel roles that are also essential to the cell. In particular, we have focused upon a yeast mitochondrial group I intron splicing reaction that requires LeuRS. The LeuRS works in collaboration with a second protein called the bI4 maturase to aid the bI4 intron splicing reaction. We have determined that the CP1 editing domain of LeuRS can also function as an independent splicing factor and possibly suggests that some determinants for amino acid editing and splicing might overlap. We are currently investigating and characterizing specific regions and also amino acids that influence the LeuRS-dependent splicing reaction.