Abstract
The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.
Highlights
Genetic code expansion is the most powerful method to introduce artificial moieties into proteins site- in live cells
As A56 is engaged in a tertiary Watson-Crick pair by U19 in the D-loop (Figure 1A), we explored the mutation U19G, both combined to A56C and on its own (Supplementary Table S1)
We reported here the generation of two enhanced orthogonal tRNAs, tRNAM15 and tRNAC15, for the Pyl orthogonal system, which is the most popular tool for non-canonical amino acids (ncAAs) incorporation into proteins expressed in mammalian cells
Summary
Genetic code expansion is the most powerful method to introduce artificial moieties into proteins site- in live cells. Non-canonical amino acids (ncAAs) are incorporated in response to an in-frame amber stop codon (UAG) by a suppressor tRNA (tRNACUA), which is charged with the ncAA by an engineered amino-acyl-tRNA-synthetase (AARS). AARS/tRNA pairs evolved for ncAA mutagenesis in mammalian cells are mostly derived from the archaeal pair that naturally incorporates pyrrolysine (Pyl) in Methanosarcina species (PylRS/tRNAPyl). While non-canonical AARS/tRNA pairs must not cross talk with the translational pairs of the host cell (criterion of orthogonality), the tRNA must be compatible with the endogenous transcription and processing machinery. Prokaryotic tRNAs that lack A-box and B-box internal promoter sequences [3], as in the case of tRNAPyl, are transcribed by placing the tRNA gene under control of RNA Pol III external promoters, such as the H1 [4] or U6 promoter [5]. Once the mature tRNA is acylated by the cognate AARS, the AA-tRNA is bound by the elongation factor, which protects it from hydrolysis and delivers it to the ribosome, where it participates in protein synthesis [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
