The parent project for this FIRCA application studies the interaction of antibiotics with ribosomal RNA. The project is aimed to elucidate the mechanisms of action of several inhibitors of translation, mechanisms of resistance to peptidyl transferase-targeted antibiotics and shed light on the function of ribosomal RNA. The major goal of the collaborative study is to identify nucleotides in the 23S ribosomal RNA peptidyl transferase center that interact with the conserved CA 3' end of transfer RNA. The authors are planning to prepare a library of randomly mutated 23S rRNA, select the mutants that are capable of forming ribosomes and select the ribosomal mutants that are capable of interacting with the mutated CA 3' end of tRNA. For the selection of the ribosomal mutant, the investigators are planning to develop peptidyl tRNA-based affinity carriers using the attachment of biotin to the anticodon end of peptidyl tRNAs and immobilizing the resulting tRNA derivatives on an avidin column. The proposal implies the development of a novel procedure of isolation of mutant ribosomes from the wild type ribosomes that are always present in the cells due to transcription from chromosomal rRNA operons. The ribosomes with mutated 23S RNA will be used not only in the proposed work, but also for the study of the rRNA interactions with antibiotics in the PI s laboratory.
