Evolutionary Medicine and Informatics: mRNA Display
Figure 1. mRNA Display. a) Each protein is covalently bound to its encoding RNA message by a DNA-puromycin linker. B) Puro-mycin is a small molecule mimic of tyrosyl tRNA. C) The peptidyl-transferase activity of the ribosome leads to the formation of an amide bond between the peptide and mRNA.
mRNA-protein fusion molecules are generated by transcribing dsDNA into ssRNA, ligating a special DNA-puromycin linker to the 3? end of the RNA, and translating the RNA template in vitro (Fig. 1a). The key to fusion formation is the puromycin molecule (Fig. 1b) attached to the 3? end of the RNA template, which mimics the structural features of amino acylated tyrosyl tRNA. During translation, the natural peptidyl transferase activity of the ribosome mediates the formation of a covalent amide bond between the 3? end of the RNA and the carboxy-terminus of the polypeptide chain. While the precise mechanistic details are not fully known, it is believed that the ribosome stalls upon reaching the DNA linker (Fig. 1c), enabling puromycin to enter the A-site and form a covalent bond with the polypeptide chain in the adjoining P-site. Thus, puromycin acts as a molecular “Rosetta Stone” by allowing previously unreadable information present in the polypeptide sequence to be read through the genetic code of the attached RNA message.