Figure 3.

“Posttranslational proofreading” allows monitoring site-specific ncAA incorporation [89]. A ubiquitin-GFP fusion reporter is encoded from the genome, while the necessary enzymes UBP1 and ClpS are overexpressed from a plasmid. The fusion reporter is synthesized via nonsense suppression where different BipRS variants acylate either biphenylalanine (BipA) (star) or a natural AA (hexagons). Ubiquitin-GFP fusions are substrates for UBP1, which removes ubiquitin (Ub) and exposes N-terminally incorporated BipA or natural AA. Truncated fusion proteins that contain destabilizing tyrosine (Tyr), Phe or Leu residues in place of the amber (TAG) stop codon are directed for degradation by ClpS. In contrast, GFP fusions containing BipA are stable and not degraded (see text for details).