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

flow cytometry;fluorescence detection;homochirality;protein synthesis;protein-labeling;puromycin;stereochemistry of translation;translation

Starck, Shelley Ruth ; Roberts, Richard W.

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: flow cytometry;    fluorescence detection;    homochirality;    protein synthesis;    protein-labeling;    puromycin;    stereochemistry of translation;    translation;   

Others  :  https://thesis.library.caltech.edu/2355/1/Abstract.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

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