The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.
This invention was made with Government support under Grant Number 2R01GM079238, 1R01GM65050 awarded by National Institute of Health; National Cancer Institute grant CA92584; U.S. Department of Energy DE-AC0376SF00098; National Institutes of Health Medical Scientist Training Program grant GM07739; National Institute of Health NRSA fellowship 1F31DC012026-01. The United States Government has certain rights in the invention.