The aminoacyl-tRNA synthetases (aaRSs) comprise a family of twenty enzymes that are essential to every living organism. Each enzyme recognizes a single cognate amino acid and covalently attaches it to the correct tRNA. The """"""""charged"""""""" tRNA then transfers the amino acid at the ribosome for specific incorporation into the growing polypeptide chain. The fidelity of protein synthesis is completely dependent on accurate substrate recognition by the aaRSs. Some aaRSs have developed editing mechanisms to correct misactivated amino acids. These editing aaRSs clear the wrong amino acid by hydrolysis of either of two substrates-misactivated aminoacyl-adenylates (""""""""pre-transfer"""""""" of arnino acid to tRNA) or misacylated aa- tRNA (""""""""post-transfer""""""""). Although one of these mechanisms may dominate, most aaRSs that edit appear to operate by a mixture of pre-and post-transfer editing, which complicates investigations to determine their respective molecular basis. E. coli leucyl-tRNA synthetase (LeuRS) is unique because it edits exclusively by a post-transfer mechanism. In the past funding cycle, the post-transfer editing activity was abolished and a pre-transfer editing pathway activated in E. coli LeuRS by a limited number of mutations. Thus, the E. coli wild-type and mutant LeuRS provide a powerful model to segregate the two aaRS fidelity mechanisms and characterize molecular determinants that are specific to pre- and/or post-transfer editing. This proposal outlines an interdisciplinary research plan that combines X-ray crystallography, computational, biochemical, and molecular biology approaches to investigate translocation mechanisms for misactivated aminoacyl- adenylate intermediates in pre-transfer editing and mischarged tRNAs in post-transfer editing. It will also determine the physiological impact of the aaRSs on translational fidelity and cell viability. A detailed understanding of editing mechanisms will benefit ongoing pharmaceutical research that capitalizes upon aaRSs as targets for antibiotic development. It will also enable re-engineering of aaRSs to activate alternate amino acids for incorporation into custom-designed proteins. These novel proteins could be used as therapeutics or important tools in medicinal and technological applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063789-07
Application #
7591051
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Bender, Michael T
Project Start
2001-09-01
Project End
2011-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
7
Fiscal Year
2009
Total Cost
$272,731
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Zhao, Hanchao; Palencia, Andres; Seiradake, Elena et al. (2015) Analysis of the Resistance Mechanism of a Benzoxaborole Inhibitor Reveals Insight into the Leucyl-tRNA Synthetase Editing Mechanism. ACS Chem Biol 10:2277-85
Pang, Yan Ling Joy; Poruri, Kiranmai; Martinis, Susan A (2014) tRNA synthetase: tRNA aminoacylation and beyond. Wiley Interdiscip Rev RNA 5:461-80
Li, Li; Martinis, Susan A; Luthey-Schulten, Zaida (2013) Capture and quality control mechanisms for adenosine-5'-triphosphate binding. J Am Chem Soc 135:6047-55
Li, Li; Palencia, Andrés; Lukk, Tiit et al. (2013) Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens. Proc Natl Acad Sci U S A 110:3817-22
Sarkar, Jaya; Poruri, Kiranmai; Boniecki, Michal T et al. (2012) Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing. J Biol Chem 287:14772-81
Palencia, Andres; Crepin, Thibaut; Vu, Michael T et al. (2012) Structural dynamics of the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase. Nat Struct Mol Biol 19:677-84
Boniecki, Michal T; Martinis, Susan A (2012) Coordination of tRNA synthetase active sites for chemical fidelity. J Biol Chem 287:11285-9
Sarkar, Jaya; Martinis, Susan A (2011) Amino-acid-dependent shift in tRNA synthetase editing mechanisms. J Am Chem Soc 133:18510-3
Sarkar, Jaya; Mao, Weimin; Lincecum Jr, Tommie L et al. (2011) Characterization of benzoxaborole-based antifungal resistance mutations demonstrates that editing depends on electrostatic stabilization of the leucyl-tRNA synthetase editing cap. FEBS Lett 585:2986-91
Li, Li; Boniecki, Michal T; Jaffe, Jacob D et al. (2011) Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites. Proc Natl Acad Sci U S A 108:9378-83

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