Protein Variants | Comment | Organism |
---|---|---|
Y349F | 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. Low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Generation of 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. They allow efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Different combinations of recognition motifs for PylRS are introduced into Bos taurus mt-tRNASerCUA. Bicistronic plasmids are generated combining each tRNA with the PylRS from Methanosarcina barkeri, which bears the mutation Y349F for enhanced activity (PylRSF). In the presence of the PylRS substrate Lys(Boc), four tRNAs in the M series and five in the C series yield higher amber suppression compared to the tRNAPyl, whereas the other tRNAs show either lower efficiency or no amber suppression at all, overview | Methanosarcina barkeri |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Methanosarcina barkeri |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-pyrrolysine + tRNAPyl | Methanosarcina barkeri | - |
AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanosarcina barkeri | Q6WRH6 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + L-pyrrolysine + tRNAPyl | - |
Methanosarcina barkeri | AMP + diphosphate + L-pyrrolysyl-tRNAPyl | - |
? | |
additional information | the archaeal tRNAPyl features a secondary structure that significantly diverges from that of canonical tRNAs | Methanosarcina barkeri | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PylRS | - |
Methanosarcina barkeri |
pyrrolysyl-tRNA synthetase | - |
Methanosarcina barkeri |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Methanosarcina barkeri |