The long term goals of this research include altering the 23S RNA in ribosomes to enable the in vitro preparation of proteins containing a broad variety of unnatural amino acids other than alpha-L-amino acids in quantities sufficient for routine biochemical studies. This will be done employing a novel selection strategy that uses modified derivatives of puromycin, a structural mimic of the 3'-end of aminoacyl-tRNA which terminates (cellular) protein synthesis in prokaryotes, resulting in cell death. By screening a large library of modified ribosomes, each of which has been expressed in E. coli following cell transformation with a (modified) plasmid-born 23S ribosomal RNA, for sensitivity to puromycin analogues containing a specific, structurally modified amino acid, it is possible to identify ribosomes potentially capable of recognizing (and incorporating) those same amino acids attached to transfer RNAs. The enhanced incorporation of beta amino acids has been achieved in this fashion. For the five-year period of requested support, the 23S rRNA constituent of ribosomes will be altered to permit the incorporation into proteins of several types of modified amino acids that native ribosomes cannot incorporate. This includes a variety of beta amino acids, which will allow definition of the specific positions/stereochemistry of substituents tolerated by the modified ribosomes. Also studied will be the ability of the beta amino acids to stabilize secondary structures in proteins.
The present project will provide access to modified ribosomes capable of incorporating very unusual structurally modified amino acids into proteins, and will do so using an innovative new strategy. It will also enable novel insights into the molecular mechanism of ribosomally mediated protein synthesis, and possibly into ways to change the mechanism. It will permit novel biomaterials to be prepared, having broad application to the analysis and treatment of diseases, including cancer and conditions associated with aging.
