{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/87898"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/87898","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Characterization of Leucyl -Trna Synthetase From Homo Sapiens and Escherichia Coli in Aminoacylation, Amino Acid Editing and Interdomain Interactions","abstract":"\"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.\"","abstract_html":""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."","abstract_has_math":false,"creators":["Pang, Yan Ling Joy"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Martinis, Susan A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-09-28T22:10:55Z","date_published":"2015-09-28T22:10:55Z","updated_at":"2026-07-22T22:26:31Z","subjects":["Chemistry, Biochemistry"],"languages":["eng"],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(MiAaPQ)AAI3431034"],"render_values":[{"text":"(MiAaPQ)AAI3431034","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/87898","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Martinis, Susan A."]},{"key":"dc:creator","label":"Author","values":["Pang, Yan Ling Joy"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-09-28T22:10:55Z","10000-01-01","2010"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"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":["Chemistry, Biochemistry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/87898","(MiAaPQ)AAI3431034"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["\"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.\"","Made available in DSpace on 2015-09-28T22:10:55Z (GMT). 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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.\"","Made available in DSpace on 2015-09-28T22:10:55Z (GMT). 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