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. doi:10.7907/WKXS-KM18. https://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

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