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

Banerjee, Aditi

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

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

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

Copyright and License Information

Copyright 2018 Aditi Banerjee

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