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Title:Leucyl-tRNA synthetase: dynamic subcellular relocalization and drug resistance mechanism
Author(s):Zhao, Hanchao
Director of Research:Martinis, Susan A.
Doctoral Committee Chair(s):Martinis, Susan A.
Doctoral Committee Member(s):Luthey-Schulten, Zaida A.; Huang, Raven H.; Jin, Hong
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):E. coli
leucyl-tRNA synthetase
CP1 domain
subcellular relocalization
periplasmic space
Abstract:The family of aminoacyl-tRNA synthetases (aaRS) are essential to all living cells. They are fundamental to setting the genetic code during protein synthesis by charging tRNA with a specific amino acid. As such, they have been selected by the pharmaceutical industries as optimal targets. A new class of antimicrobial benzoxaborole compounds was identified as a potent inhibitor of leucyl-tRNA synthetase (LeuRS) and therefore of protein synthesis. AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) blocks fungal cytoplasmic LeuRS by trapping tRNALeu in the editing site of the enzyme’s CP1 domain. Some resistant mutations are located outside of the CP1 hydrolytic editing active site. Thus, their mode of action was not understood. A combination of X-ray crystallography, molecular dynamics, biochemical experiments, and mutational analysis of a distal benzoxaborole-resistant mutant uncovered a eukaryote-specific tyrosine “switch” that is critical to tRNA-dependent post-transfer editing. The tyrosine "switch" has three states that shift between interactions with a lysine and the 3’-hydroxyl of the tRNA terminus, to inhibit or promote post-transfer editing. The benzoxaborole’s mechanism of action capitalizes upon one of these editing active site states. Evolution of this tunable editing mechanism in eukaryotic and archaeal LeuRSs is proposed to enable precise control of aminoacylation fidelity. The aaRSs have also been adapted through evolution for alternate functions that are entirely distinct from proteins synthesis. In E. coli, small fractions of LeuRS were identified outside the cytoplasm, in the periplasmic space and also associated with the membrane. Under certain stresses, LeuRS and tRNALeu re-located into the media. In some cases, LeuRS appeared to be fragmented with one part associated with the membrane, and a second part in the periplasmic space. It is hypothesized that E. coli LeuRS plays a non-canonical role as a leucine sensor and cell signaling akin to yeast and mammalian cells.
Issue Date:2016-01-20
Rights Information:Copyright 2016 Hanchao Zhao
Date Available in IDEALS:2016-07-07
Date Deposited:2016-05

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