The role of nonstandard purine and amino acid moieties of puromycin in ribosome substrate binding

Peter C. Anthony
Yale University

The antibiotic puromycin mimics the 3’ terminus of an aminoacylated tRNA and causes non-specific, premature termination of translation. The molecule consists of N6-dimethyl adenosine joined to O-methyl tyrosine by a 3’ amide linkage. In the ribosome, puromycin enters the A-site and accepts the nascent polypeptide, but following translocation, the polypeptide cannot be elongated further because puromycin’s amide linkage is unsusceptible to nucleophilic attack by the next charged tRNA. It is currently unclear what purposes are served by puromycin’s other nonstandard chemical moieties—the dimethyl amine on the purine and the amino acid sidechain. We are using biochemical methods to compare interactions of puromycin and N-acetylated phenylalanyl adenosine with rRNA bases near the site of peptidyl transfer in large ribosomal subunits. From puromycin and adenosine, we have used solution- and solid-phase techniques to synthesize trinucleotides that differ only by the presence or absence of N6-methyl and O-methyl groups on the 3’ residue. Preliminary CMCT footprinting of molecules with tBOC-protected peptidyl amines shows no statistically significant difference in protection of the E. coli P-site base U2585. Work is ongoing to identify and characterize interactions with other bases in the A- and P-sites in E. coli and H. marismortui ribosomes.