The long term goals of this research involve modification of the peptidyltransferase (PTC) center in bacterial ribosomes to enable the incorporation of very unusual amino acids not recognized by wild- type ribosomes. In recent years, we have described the selection of modified ribosomes and their use in incorporating D-amino acids, beta amino acids, dipeptides and dipeptidomimetric cassettes into proteins. During the four years of proposed research, the focus of efforts will be on the selection of ribosomes capable of introducing phosphorylated and glycosylated amino acids into specific proteins. The utility of this approach will be explored by analyzing the altered function of proteins into which phosphates and carbohydrates have been introduced, and by expressing a phosphorylated DNA binding protein in E. coli.
The present project is aimed at employing modified bacterial ribosomes to incorporate phosphorylated and glycosylated amino acids into specific proteins. These modified proteins will have their properties altered by the introduction of phosphate and sugar moieties, enabling protein behavior to be better understood by the use of substrates of defined structure in systems that rely on the presence of functional groups usually added posttranslationally. Also proposed is the in cellulo introduction of phosphotyrosine into a DNA binding protein.
