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Nucleic Acids Research, 2000, Vol. 28, No. 5 1176-1182
© 2000 Oxford University Press

Specific bonding of puromycin to full-length protein at the C-terminus

Etsuko Miyamoto-Sato1,2, Naoto Nemoto1, Kensei Kobayashi2 and Hiroshi Yanagawa1,*

1Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan and 2Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan

Puromycin, an analog of the 3' end of aminoacyl-tRNA, causes premature termination of translation by being linked non-specifically to growing polypeptide chains. Here we report the interesting phenomenon that puromycin acting as a non-inhibitor at very low concentration (e.g. 0.04 µM) can bond only to full-length protein at the C-terminus. This was proved by using a carboxypeptidase digestion assay of the products obtained by Escherichia coli cell-free translation of human tau 4 repeat (tau4R) mRNA in the presence of low concentrations of puromycin or its derivatives. The tau4R mRNA was modified to code for three C-terminal methionines, which were radioactively labeled, followed by a stop codon. The translation products could not be digested by carboxy-peptidase if puromycin or a derivative was present at the C-terminus of full-length tau4R. Puromycin and its derivatives at 0.04–1.0 µM bonded to 7–21% of full-length tau4R, depending on the ability to act as acceptor substrates. Furthermore, the bonding efficiency of a puromycin derivative to tau4R was decreased by addition of release factors. These results suggest that puromycin and its derivatives at concentrations lower than those able to compete effectively with aminoacyl-tRNA can bond specifically to full-length protein at a stop codon. This specific bonding of puromycin to full-length protein should be useful for in vitro selection of proteins and for in vitro and in vivo C-terminal end protein labeling.

* To whom correspondence should be addressed. Tel: +81 427 24 6293; Fax: +81 427 24 6317; Email: hyana@libra.ls.m-kagaku.co.jp


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