{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/102832"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/102832","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Editing by leucyl-trna synthetase: Discrimination of norvaline and isoleucine","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.","abstract_html":"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.","abstract_has_math":false,"creators":["Banerjee, Aditi"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Martinis, Susan A.","van der Donk, Wilfred A.","Luthey-Schulten, Zaida A.","Jin, Hong"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-02-07T20:44:09Z","date_published":"2019-02-07T20:44:09Z","updated_at":"2026-07-22T22:24:42Z","subjects":["Aminoacyl tRNA synthetase, tRNA, Leucyl tRNA synthetase, Leucine, Isoleucine, Norvaline"],"languages":["en"],"rights":["Copyright 2018 Aditi Banerjee"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/102832","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Martinis, Susan A.","van der Donk, Wilfred A.","Luthey-Schulten, Zaida A.","Jin, Hong"]},{"key":"dc:creator","label":"Author","values":["Banerjee, Aditi"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-02-07T20:44:09Z","2021-02-08T10:15:29Z","2018-12-05","2018-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Aminoacyl tRNA synthetase, tRNA, Leucyl tRNA synthetase, Leucine, Isoleucine, Norvaline"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2018 Aditi Banerjee"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/102832"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2020-12-01","The student, Aditi Banerjee, accepted the attached license on 2018-12-04 at 09:13.","The student, Aditi Banerjee, submitted this Dissertation for approval on 2018-12-04 at 09:24.","This Dissertation was approved for publication on 2018-12-05 at 08:43.","DSpace SAF Submission Ingestion Package generated from Vireo submission #13192 on 2019-02-07 at 14:18:57","Made available in DSpace on 2019-02-07T20:44:09Z (GMT). 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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.","Submission published under a 24 month embargo labeled 'U of I Access', the embargo will last until 2020-12-01","The student, Aditi Banerjee, accepted the attached license on 2018-12-04 at 09:13.","The student, Aditi Banerjee, submitted this Dissertation for approval on 2018-12-04 at 09:24.","This Dissertation was approved for publication on 2018-12-05 at 08:43.","DSpace SAF Submission Ingestion Package generated from Vireo submission #13192 on 2019-02-07 at 14:18:57","Made available in DSpace on 2019-02-07T20:44:09Z (GMT). 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