PMID:10666460
| Citation |
Miyamoto-Sato, E, Nemoto, N, Kobayashi, K and Yanagawa, H (2000) Specific bonding of puromycin to full-length protein at the C-terminus. Nucleic Acids Res. 28:1176-82 |
|---|---|
| Abstract |
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 microM) 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 microM 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Binding Sites; Cell-Free System; Escherichia coli; Humans; Molecular Sequence Data; Protein Binding; Proteins/chemistry; Proteins/metabolism; Puromycin/chemistry; Puromycin/metabolism |
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