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Exploring the proteome: insights into eukaryotic protein synthesis using puromycin analogs

Citation

Starck, Shelley Ruth (2004) Exploring the proteome: insights into eukaryotic protein synthesis using puromycin analogs. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06012004-235945

Abstract

Puromycin is a protein synthesis inhibitor that acts as a structural analog of an aminoacyl-tRNA. The ribosome mistakenly inserts puromycin in place of aminoacyl-tRNA resulting in truncated proteins containing the drug at their C-terminus. Herein, the puromycin reaction is re-examined in a cell-free eukaryotic translation extract and in live cells. The framework for puromycin reactivity in terms of potency, product distribution, and mechanism is studied in vitro. These insights are used to develop a series of fluorescent puromycin-based reagents to detect protein expression in living cells that does not require transfection, radiolabeling, or the prior choice of a candidate gene. Further, puromycin is used to examine the stereo- and regiochemical nature of protein synthesis both in vitro and in vivo. These data indicate that the ribosome tolerates a broader range of substrates than previously recognized, including D-amino acids. Overall, these studies yield a series of insights about protein synthesis and the ribosome, including mechanistic observations, the development of reagents to explore the proteome, and a theory about the evolution of amino acid homochirality

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:flow cytometry; fluorescence detection; homochirality; protein synthesis; protein-labeling; puromycin; stereochemistry of translation; translation
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Roberts, Richard W.
Thesis Committee:
  • Dougherty, Dennis A. (chair)
  • Gray, Harry B.
  • Dervan, Peter B.
  • Roberts, Richard W.
Defense Date:20 May 2004
Author Email:srstarck (AT) caltech.edu
Record Number:CaltechETD:etd-06012004-235945
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06012004-235945
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2355
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:03 Jun 2004
Last Modified:26 Dec 2012 02:50

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