University of Illinois Urbana-Champaign

Editing by leucyl-trna synthetase: Discrimination of norvaline and isoleucine

Banerjee, Aditi

Content Files
BANERJEE-DISSERTATION-2018.pdf

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https://hdl.handle.net/2142/102832

Description

Title
Editing by leucyl-trna synthetase: Discrimination of norvaline and isoleucine
Author(s)
Banerjee, Aditi
Issue Date
2018-12-05
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.
  • Luthey-Schulten, Zaida A.
  • Jin, Hong
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2019-02-07T20:44:09Z
Keyword(s)
Aminoacyl tRNA synthetase, tRNA, Leucyl tRNA synthetase, Leucine, Isoleucine, Norvaline
Abstract
Aminoacyl tRNA-synthetases (AARS) are housekeeping enzymes that are tasked with accurate synthesis of aminoacylated tRNA for protein synthesis and other cellular functions. The specificity of amino acid attachment challenges the AARSs that need to distinguish between structurally similar amino acids. In such cases, AARSs have developed editing mechanisms to circumvent the issue of misaminoacylation. Leucyl-tRNA synthetase (LeuRS), for instance selectively edits misactivated and mischarged non-leucine amino acids via pre-transfer editing of misactivated adenylates in the synthetic site or by hydrolyzing mischarged amino acids in the CP1 editing domain. The enzyme’s dependence between the two editing mechanisms can shift based on the origin from which the AARS is derived, the amino acid that is targeted for editing, or presence of a mutation in the enzyme. In the absence of the CP1 domain, E. coli LeuRS (LeuRS-ΔCP1) maintains fidelity by clearing non-leucine aminoacyl-adenylates in the enzyme’s synthetic site. The intact tRNA 3’-terminal adenosine (A76) residue is a prerequisite for aminoacylation. Leveraging A76 essentiality tRNA analogues were designed to investigate amino acid dependent specificity of editing by LeuRS. The tRNA analogues were synthesized by addition of a modified adenosine triphosphate to an in vitro transcribed E. coli tRNALeuUAA using the CCA-adding enzyme from E. coli. Incorporation of unchargeable tRNA analogues stimulated ATP hydrolysis by wild type LeuRS in the presence of norvaline. In contrast, pre-transfer editing occurs independent of the tRNA for LeuRS-ΔCP1, which lacks the CP1 domain. Therefore it is hypothesized that the CP1 domain of LeuRS plays a critical role for tRNA-dependent pre-transfer editing.
Graduation Semester
2018-12
Type of Resource
text
Permalink
http://hdl.handle.net/2142/102832
Copyright and License Information
Copyright 2018 Aditi Banerjee

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