Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery.

Stop codon suppression using dedicated tRNA/aminoacyl-tRNA synthetase (aaRS) pairs allows for genetically encoded, site-specific incorporation of non-canonical amino acids (ncAAs) as chemical handles for protein labeling and modification. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal pyrrolysine tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual-fluorophore labeling of a cell surface receptor on live mammalian cells.

Electrostatic interactions guide substrate recognition of the prokaryotic ubiquitin-like protein ligase PafA.

Pupylation, a post-translational modification found in Mycobacterium tuberculosis and other Actinobacteria, involves the covalent attachment of prokaryotic ubiquitin-like protein (Pup) to lysines on target proteins by the ligase PafA (proteasome accessory factor A). Pupylated proteins, like ubiquitinated proteins in eukaryotes, are recruited for proteasomal degradation. Proteomic studies suggest that hundreds of potential pupylation targets are modified by the sole existing ligase PafA. This raises intriguing questions regarding the selectivity of this enzyme towards a diverse range of substrates. Here, we show that the availability of surface lysines alone is not sufficient for interaction between PafA and target proteins. By identifying the interacting residues at the pupylation site, we demonstrate that PafA recognizes authentic substrates via a structural recognition motif centered around exposed lysines. Through a combination of computational analysis, examination of available structures and pupylated proteomes, and biochemical experiments, we elucidate the mechanism by which PafA achieves recognition of a wide array of substrates while retaining selective protein turnover.