Initiation of Protein Synthesis with Non-Canonical Amino Acids In?Vivo |
| |
| Authors: | Dr Jeffery M Tharp Dr Omer Ad Dr Kazuaki Amikura Fred R Ward Emma M Garcia Prof?Dr Jamie H D Cate Prof?Dr Alanna Schepartz Prof?Dr Dieter Söll |
| |
| Institution: | 1. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06520 USA;2. Department of Chemistry, Yale University, New Haven, CT, 06520 USA;3. Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720 USA |
| |
| Abstract: | By transplanting identity elements into E. coli tRNAfMet, we have engineered an orthogonal initiator tRNA (itRNATy2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNATy2 can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNAfMet from the genome afforded an E. coli strain in which the efficiency of non-canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo. |
| |
| Keywords: | Chemische Biologie Erweiterung des genetischen Codes Nicht-kanonische Aminosäuren Synthetische Biologie Translationsinitiation |
|
|
