Mechanisms underlying the specificity of aminoacyl-tRNA synthesis will be studied in the glutamyl and glutaminyl systems. First, E. coli glutaminyl-tRNA synthetase will be used as a model system to break new ground in our understanding of intramolecular communication between the amino acid and tRNA binding sites of tRNA synthetases. Alanine scanning mutagenesis will be used to create a mapping of which enzyme-tRNA contacts influence the affinity for glutamine. This overall study will then be followed with an in-depth analysis of the roles of indirect readout, electrostatics, and conformational strain in communication between the tRNA acceptor stem interface and the adjacent glutamine binding site. Next, we will use a novel chemical and enzymatic synthesis approach to study the importance of the 2-thiouridine modification at the anticodon wobble base position, in both the glutaminyl and glutamyl systems. Further studies are proposed on a diverse set of glutamyl- tRNA synthetases, to uncover the structural and energetic basis for relaxation of tRNA specificity in non-discriminating enzymes. Finally, further studies on an engineered hybrid GlnRS enzyme with significant capacity to synthesize glutamylated tRNAGln are proposed. Throughout the application, a common theme is the integration of pre-steady state kinetic measurements with X-ray structural data. A new theme is the further inclusion of in silico bioinformatics approaches to derive correlations from the exhaustive tRNA and tRNA synthetase databases. The elucidation of how induced fit and indirect readout mechanisms control tRNA and amino acid specificities in these systems will be relevant to understanding such processes in more complex particles such as the ribosome. Further, there is great potential for developing novel antimicrobial compounds based on the discrimination between human and bacterial synthetases. Finally, engineering of tRNA synthetases to expand the genetic code may open the door to novel protein-based therapeutics and has potential to impact the development of therapies for many human diseases.

Public Health Relevance

Aminoacyl-tRNA synthetases are essential enzymes in every living cell. Understanding how they function in protein synthesis is crucial to public health because many human diseases can be traced to defective proteins. In addition to this very general role in synthesizing all proteins, one aminoacyl-tRNA synthetase studied in this project is also involved in regulating the expression of genes that control inflammation in humans. This enzyme, the human glutamyl-tRNA synthetase, thus has a particular known relevance to public health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063713-13
Application #
8269728
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Barski, Oleg
Project Start
2001-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
13
Fiscal Year
2012
Total Cost
$289,864
Indirect Cost
$84,138
Name
Portland State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
052226800
City
Portland
State
OR
Country
United States
Zip Code
97207
Perona, John J; Gruic-Sovulj, Ita (2014) Synthetic and editing mechanisms of aminoacyl-tRNA synthetases. Top Curr Chem 344:1-41
Dulic, Morana; Perona, John J; Gruic-Sovulj, Ita (2014) Determinants for tRNA-dependent pretransfer editing in the synthetic site of isoleucyl-tRNA synthetase. Biochemistry 53:6189-98
Bhaskaran, Hari; Taniguchi, Takaaki; Suzuki, Takeo et al. (2014) Structural dynamics of a mitochondrial tRNA possessing weak thermodynamic stability. Biochemistry 53:1456-65
Hadd, Andrew; Perona, John J (2014) Coevolution of specificity determinants in eukaryotic glutamyl- and glutaminyl-tRNA synthetases. J Mol Biol 426:3619-33
Rodriguez-Hernandez, Annia; Spears, Jessica L; Gaston, Kirk W et al. (2013) Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position. J Mol Biol 425:3888-906
Oza, Javin P; Sowers, Kevin R; Perona, John J (2012) Linking energy production and protein synthesis in hydrogenotrophic methanogens. Biochemistry 51:2378-89
Bhaskaran, Hari; Rodriguez-Hernandez, Annia; Perona, John J (2012) Kinetics of tRNA folding monitored by aminoacylation. RNA 18:569-80
Grant, Thomas D; Snell, Edward H; Luft, Joseph R et al. (2012) Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase. Nucleic Acids Res 40:3723-31
Bhaskaran, Hari; Perona, John J (2011) Two-step aminoacylation of tRNA without channeling in Archaea. J Mol Biol 411:854-69
Rodriguez-Hernandez, Annia; Perona, John J (2011) Heat maps for intramolecular communication in an RNP enzyme encoding glutamine. Structure 19:386-96

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