Making sense out of nonsense

There is considerable interest among protein scientists in methods that permit unnatural amino acids to be incorporated at specific sites in proteins. Such methods would facilitate studies of protein structure and function and would allow for the synthesis of proteins having novel properties. Current methods use nonsense-suppressing tRNAs (1) to incorporate an unnatural amino acid at the desired site in a protein and chemical (rather than enzymatic) means to attach the unnatural amino acid to the nonsense-suppressing tRNA (2). However, protein yields are typically modest because the suppressor tRNA participates in only one round of translation. Protein expression could be increased by the development of in vivo systems, but this poses significant challenges. It requires a synthetase that activates an unnatural amino acid and only attaches it to a designated tRNA. In addition, it requires that the designated tRNA is not aminoacylated by any other synthetase in the cell and that it efficiently translates a nonsense codon. A “21st cognate pair” is essentially created if all of these criteria are met (Fig. 1). The paper by Kowal et al. (3) in this issue of PNAS makes an important contribution by describing a novel approach for creating tRNA-synthetase pairs that efficiently incorporate natural amino acids at specific sites in proteins in vivo. By taking a novel approach in their choice of tRNAs and synthetases for membership in a new cognate pair, RajBhandary's group has created two in vivo systems that substantially alleviate the protein expression problem. Network of potential interactions among tRNAs (cloverleaf) and synthetases (“C-shape”). (A) Hypothetical wild-type case having three cognate pairs. In the typical cell, about 1,200 potential interactions are possible. (B) The increase in network complexity that arises when a new cognate pair is introduced. Colored arrows indicate cognate interactions, and dashed arrows indicate interactions that …