Crystallographic Studies of Ribosome Functional Sites
A system will be developed that allows insertion of mutations in the 23S rRNA and produces high quality crystals of the 50S ribosomal subunit. Such a system will provide the means to study the structural effects of mutations at atomic resolution. For example, the structural effects of guanine to adenine mutations at position 2482 (Haloarcula marismortui) in rRNA will be studied crystallographically. Position 2482 is structurally important at the peptidyl transferase active site, and guanine to adenine mutations at this site confers resistance to chloramphenicol antibiotic. Structural information about mutations at G2482 will contribute to understanding the mechanisms of the peptidyl transferase reaction and antibiotic action on ribosomes. The binding site of evernimicin, a new experimental antibiotic used to treat multidrug-resistant bacterial infections, will be studied by soaking the drug into crystals of H marismortui 50S ribosomal subunits. Structural information about the binding site of this drug may contribute to understanding this novel mechanism of antibiotic action on ribosomes. The effects of ionic strength and formation of base triples on the pKa of adenines will be studied using optical melting and NMR. Because adenines are functionally important in the active site of the 50S ribosomal subunit and other ribozymes, a better understanding of the structural contexts in which the pKa of adenines changes will contribute to understanding structural-function relationships in RNA.