pyrrolysyltRNA

Pyrrolysyl-tRNA, typically referred to as tRNA^Pyl, is a transfer RNA that delivers the amino acid pyrrolysine (Pyl) to the ribosome during translation. It functions together with the enzyme pyrrolysyl-tRNA synthetase (PylS), which charges tRNA^Pyl with pyrrolysine. The pyrrolysine-incorporation system is found in a subset of methanogenic archaea and, in some cases, bacteria, where tRNA^Pyl carries an anticodon that recognizes the amber stop codon UAG, enabling pyrrolysine insertion at sites specified by this codon rather than termination. The genetic module typically includes pylS, pylT (the gene encoding tRNA^Pyl), and the pylB/C/D cluster involved in pyrrolysine biosynthesis.

Biochemically, tRNA^Pyl is recognized by PylS, which attaches pyrrolysine to the 3’ end of the tRNA. In pyl-positive organisms, pyrrolysine is derived from lysine through a biosynthetic pathway encoded by pylB/C/D, allowing cellular production of the amino acid for incorporation during translation. The tRNA^Pyl anticodon is designed to pair with UAG, enabling codon reassignment in the organism where this system operates.

Applications and significance include the use of the PylS/tRNA^Pyl pair for genetic code expansion in research. Because the system is largely orthogonal to host tRNA synthetases, it can be introduced into bacteria, yeast, and mammalian cells to incorporate pyrrolysine or various noncanonical amino acids at amber codons. This enables site-specific protein labeling, the study of protein function, and the development of novel biomolecules.