University of Illinois Urbana-Champaign

Characterization of leucyl-TRNA synthetase from homo sapiens and escherichia coli in aminoacylation, amino acid editing and interdomain interactions

Pang, Yan Ling Joy

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1_Pang_YanLingJoy.pdf

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https://hdl.handle.net/2142/15542

Description

Title
Characterization of leucyl-TRNA synthetase from homo sapiens and escherichia coli in aminoacylation, amino acid editing and interdomain interactions
Author(s)
Pang, Yan Ling Joy
Issue Date
2010-05-14T20:44:47Z
Director of Research (if dissertation) or Advisor (if thesis)
Martinis, Susan A.
Doctoral Committee Chair(s)
Martinis, Susan A.
Committee Member(s)
  • van der Donk, Wilfred A.
  • Shapiro, David J.
  • Chen, Lin-Feng
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2010-05-14T20:44:47Z
Keyword(s)
  • aminoacyl-tRNA synthetases
  • Connective polypeptide 1 (CP1)
  • human cytoplasmic leucyl-tRNA synthetases
  • tRNA
  • fidelity in protein synthesis
  • translocation
  • amino acids
  • leucine
Abstract
Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that charge tRNA with its cognate amino acid. In order to maintain fidelity during protein synthesis, editing mechanisms ensure that tRNAs are accurately charged. Leucyl-tRNA synthetase (LeuRS) has an editing active site that resides in a discrete domain called the connective polypeptide 1 domain (CP1). Post-transfer editing involves the translocation of mischarged tRNA from the aminoacylation to the editing active site where mischarged tRNA binds for hydrolysis of the noncognate amino acid to enhance fidelity. Based on crystal structure analysis, the CP1 domain rotates 30° relative to the canonical core where aminoacylation occurs during tRNA translocation and presumably facilitates the movement of tRNA from the core domain to the editing domain. Single molecule fluorescence resonance energy transfer (smFRET) techniques were employed to characterize this dynamic movement of tRNA from one domain of the enzyme to another. Human cytoplasmic LeuRS (hscLeuRS) is typically found in a macromolecular complex containing at least eight other proteins. In order to study this enzyme, hscLeuRS was expressed independent of the complex in Escherichia coli. Enzymatic characterization of the isolated hscLeuRS suggested that it attaches a second leucine to Leu-tRNALeu. Liquid chromatography and mass spectrometry methods were used in an attempt to isolate this hypothesized “doubly charged” tRNA species and it is possible that hscLeuRS possesses a secondary function beyond aminoacylation reliant on a doubly charged Leu-Leu-tRNALeu. Further biochemical analysis of the hscLeuRS focused on its editing pocket. The editing site of hscLeuRS includes a highly conserved threonine discriminator and universally conserved aspartic acid that were mutationally characterized. Substitution of threonine to alanine uncoupled specificity similar to other LeuRSs. However, the introduction of bulky residues in the amino acid binding pocket failed to block deacylation of tRNA, indicating that the architecture of the amino acid binding pocket is different compared to other characterized LeuRSs. In addition, mutation of the universally conserved aspartic acid abolished tRNALeu deacylation. Surprisingly though, this editing-defective hscLeuRS maintained fidelity. This indicates that an alternate editing mechanism may have been activated upon failure of the post-transfer editing active site in order to maintain fidelity during protein synthesis.
Graduation Semester
2010-5
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http://hdl.handle.net/2142/15542
Copyright and License Information
Copyright 2010 Yan Ling Joy Pang

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