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Driving orthogonality between variant pyrrolysyl-tRNA synthetase/tRNA pairs

By Patrick Ginther

In a recent 2018 Nature news & views, the work of Jason Chin and co-workers was highlighted for the lab’s extraordinary work on developing new tools for synthetic biology [1]. Specifically, Chin and co-workers successfully generated a variant of the pyrrolysyl-tRNA synthetase/tRNA pair (PylRS/pyltRNA) derived from Methanomethylophilus alvus (Ma) that was mutually orthogonal to the commonly used Methanosarcina mazei (Mm) and methanosarcina barkeri (Mb) PylRS/pyltRNA pairs [2].

The novelty of the experiment comes from how the lab went about developing this mutually orthogonal PylRS/pyltRNA pair. Three classes of PylRS exist in nature: one in which the N-terminal and C-terminal domains are expressed as one protein, a second in which the N-terminal domain is encoded separately from the C-terminal domain, and a third class in which there is no N-terminal domain. They rationalized that since the N-terminal domain is known to encode specificity for its PyltRNA partner, selecting for a PylRS/pyltRNA pair in which the synthetase lacks an N-terminal domain all together may instill mutual orthogonality with a PylRS/pyltRNA pair which has N-terminal recognition.

Indeed, after searching databases for a synthetase with high homology to MmPylRS and eliminating any hits that contained an N-terminal domain, they identified five PylRS/pyltRNA pairs. Of those hits, the MaPylRS, had the highest non-conical amino acid (ncAA) incorporation, but was recognized by the MmPylRS. Instead of pursuing another hit that showed orthogonality at the cost of significantly lower ncAA incorporation, the lab decided to evolve an orthogonal MaPylRS/MaPyltRNA pair by mutating the variable loop region of the MaPyltRNA. Through these mutations, the lab generated a MaPyltRNA that was not recognized by MmPylRS and thus created a new PylRS/pyltRNA pair that is mutually orthogonal to existing PylRS/pyltRNA pairs.

  1. Chen, PR & Ngai WSC (2018) “Optimizing orthogonality”. Nat Chem, 10, 802-803. doi:10.1038/s41557-018-0115-7
  2. Willis JCW & Chin JW (2018) Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nat Chem, 10, 831-837. doi:10.1038/s41557-018-0052-5