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Analysis of the enzymological properties of prolyl-tRNA synthetases in plants focusing on the misactivation of the proline analog azetidine-2-carboxylic acid
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Text
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Title
Analysis of the enzymological properties of prolyl-tRNA synthetases in plants focusing on the misactivation of the proline analog azetidine-2-carboxylic acid
SubTitle
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PartNumber
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ETD_2128
Identifier
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000052259
Language
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eng
Genre
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theses
Subject
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(authority = RUETD)
Topic
Plant Biology
Subject
(ID = SBJ-2);
(authority = ETD-LCSH)
Topic
Aminoacyl-tRNA synthetases
Subject
(ID = SBJ-3);
(authority = ETD-LCSH)
Topic
Ligases
Abstract
Azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline (Pro), inhibits the growth of bacterial, plant and animal cells. Toxicity is thought to occur when A2C is incorporated into proteins in place of Pro resulting from misrecognition by prolyl-tRNA synthetases (ProRS). Arabidopsis thaliana seedlings are highly sensitive to A2C resulting in growth inhibition. To explain this result, the activity of the two forms of ProRS from A. thaliana was studied. One form of ProRS is localized to chloroplasts/mitochondria and the other form to the cytosol. Both forms were expressed as His-tagged recombinant proteins in Escherichia coli. Purified enzymes were functionally active in the ATP-PPi exchange, and aminoacylation assays demonstrated similar Km values for Pro. A major difference was observed in the specificity for A2C. The organellar form showed a 340-fold greater Km[A2C] than the cytosolic enzyme. These results suggest that A2C-sensitivity of A. thaliana is primarily due to the inability of cytosolic ProRS to distinguish between Pro and A2C. A similar result was obtained with the ProRSs from Zea mays suggesting that the difference in substrate specificity is a conserved feature of plant cytosolic and organellar ProRSs. The tRNA-specificity of the organellar and cytosolic enzymes was also examined. The organellar ProRS At5g52520 was able to complement a conditional ProRS mutant strain of E. coli but the cytosolic ProRS At3g62120 did not. At5g52520 was able to efficiently aminoacylate both E. coli and Saccharomyces cerevisiae tRNA. In contrast, At3g62120 was able to aminoacylate S. cerevisiae tRNA more efficiently than E. coli tRNA. The interest in ProRS arose initially from the observation that some plant species, including Convallaria majalis and Polygonatum multiflorum, two members of the Liliopsida, accumulate high levels of A2C. In 1972, Norris et el. reported that ProRS from C. majalis can discriminate between Pro and A2C (Norris and Fowden, 1972). To gain a better understanding of the mechanism of A2C resistance, the cDNAs encoding ProRS were isolated from C. majalis and Polygonatum pubescens. Based on sequence homology, the cDNA encoding organellar ProRS was successfully cloned. However, attempts to produce a functional ProRS from the cloned cDNAs were unsuccessful.
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electronic resource
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xiii, 186 p. : ill.
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Note
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Ph.D.
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Includes bibliographical references (p. 178-184)
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by Jiyeon Lee
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Lee
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Jiyeon
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1975-
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Jiyeon Lee
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Leustek
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Thomas
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chair
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Advisory Committee
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Thomas Leustek
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Lawton
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Michael
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internal member
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Michael A. Lawton
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Zilinskas
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Barbara
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Barbara A. Zilinskas
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Kerstetter
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Randall
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Randall A. Kerstetter
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Kahn
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Peter
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Peter C. Kahn
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Rutgers University
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Graduate School - New Brunswick
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OriginInfo
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2009
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2009-10
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xx
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Rutgers University Electronic Theses and Dissertations
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ETD
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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Identifier
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doi:10.7282/T3RN380X
Genre
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ETD doctoral
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Rights
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The author owns the copyright to this work.
Copyright
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Copyright protected
Notice
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Open
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Permission or license
Note
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Lee
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Jiyeon
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2009-09-30 23:30:01
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Jiyeon Lee
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Rutgers University. Graduate School - New Brunswick
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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365 days
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