Pyrrolysine is incorporated using a dedicated
Pyrrolysine amber suppression requires dedicated orthogonal translation components distinct from canonical termination machinery. Incorporation at in-frame UAG within methylamine methyltransferase genes depends on specialized transfer RNA tRNAPyl bearing CUA anticodon and cognate pyrrolysyl-tRNA synthetase PylRS charging tRNAPyl with pyrrolysine exclusively without interacting with host synthetases. Delivery to ribosome utilizes elongation factor EF-Tu or archaeal EF1A competing with release factor RF1. No release factor or RNA polymerase directly inserts amino acid; only translation apparatus
Ref: NCBI Bookshelf, Synthetic Biology: tRNA and Synthetase for Pyrrolysine Insertion