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Biochemical analysis of the mRNA scavenger decapping enzymes
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Title
Biochemical analysis of the mRNA scavenger decapping enzymes
Name
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Liu
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Shin-Wu
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Shin-Wu Liu
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author
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Kiledjian
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Megerditch
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Advisory Committee
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Megerditch Kiledjian
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chair
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Covey
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Lori
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Advisory Committee
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Lori Covey
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internal member
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Gunderson
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Samuel
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Advisory Committee
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Samuel Gunderson
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Kinzy
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Terri
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Advisory Committee
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Terri Kinzy
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outside member
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Patel
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Smita
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Advisory Committee
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Smita Patel
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Rutgers University
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degree grantor
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Graduate School - New Brunswick
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theses
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2007
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2007
Language
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English
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electronic
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xii, 126 pages
Abstract
The modulation of mRNA degradation is an essential determining point for regulation of gene expression. Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNAs. The scavenger mRNA decapping enzyme, DcpS, hydrolyzes the cap structure, which is the decay product in the 3'-5' mRNA degradation pathway. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m7GpppN to release m7Gp and ppN. We have carried out a biochemical characterization of the DcpS enzyme and demonstrated that, unlike other HIT family members, DcpS requires both the core HIT fold at the C-terminus and a segment of N terminus for cap binding and hydrolysis. To further examine the cellular function of DcpS, we tested the impact of DcpS on cap-dependent translation. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure in the presence of eIF4E, an essential cap-binding translation initiation factor. Furthermore, we demonstrated that the relative cap-dependent translation was inhibited by 40% in the DcpS knockdown cells and was partially restored by DcpS complementation. These results strongly suggest that DcpS functions to prevent the accumulation of residual cap structure that would otherwise trap eIF4E and interfere with cap-dependent translational events.
Structural analysis of DcpS revealed that it is a dimeric protein with a distinct N terminal domain and a C terminal domain, linked by a flexible hinge region, which led to a proposed dynamic decapping model, where the N terminus flips back and forth during the process of hydrolysis. To gain more insights into the decapping mechanism at the subunit level, we analyzed the kinetics of DcpS decapping and demonstrated that its decapping was negatively regulated under multiple turnover conditions, due to an allosteric conformational change caused by the excess amount of substrate. Our data have provided mechanistic details in terms of hydrolysis as well as the insights into the regulation of decapping in cells.
Structural analysis of DcpS revealed that it is a dimeric protein with a distinct N terminal domain and a C terminal domain, linked by a flexible hinge region, which led to a proposed dynamic decapping model, where the N terminus flips back and forth during the process of hydrolysis. To gain more insights into the decapping mechanism at the subunit level, we analyzed the kinetics of DcpS decapping and demonstrated that its decapping was negatively regulated under multiple turnover conditions, due to an allosteric conformational change caused by the excess amount of substrate. Our data have provided mechanistic details in terms of hydrolysis as well as the insights into the regulation of decapping in cells.
Note
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Ph.D.
Note
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Includes bibliographical references (p. 108-125).
Subject
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Topic
Cell and Developmental Biology
Subject
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Topic
Gene expression
Subject
(ID = SUBJ3);
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Topic
Genetic regulation
Subject
(ID = SUBJ4);
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Topic
Messenger RNA
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Graduate School - New Brunswick Electronic Theses and Dissertations
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rucore19991600001
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.16729
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ETD_551
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Identifier
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doi:10.7282/T36973ZH
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ETD doctoral
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The author owns the copyright to this work.
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Copyright protected
Availability
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Open
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Shin-Wu Liu
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Affiliation
Rutgers University. Graduate School - New Brunswick
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Non-exclusive ETD license
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Author Agreement License
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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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