NSLS-II   |   Brookhaven National Laboratory

Snapshots of Ribosome Assembly in Bacteria by Synchrotron X-ray Footprinting

Sarah Woodson, Johns Hopkins University

Folding of rRNA during ribosome assembly depends on the intrinsic stability of rRNA interactions and a hierarchy of protein-induced conformational changes that lock the complex into its final form. RNA “footprinting” using a white light beam at NSLS X28C allows changes in the RNA structure and protein interactions to be probed with millisecond resolution. Time-resolved hydroxyl radical footprinting of the rRNA during 30S assembly showed that individual proteins protect their rRNA binding sites at multiple rates, suggesting induced fit or gradual accommodation of the protein-RNA interactions. Protein S4 stabilizes most of the body of the 30S ribosome, illustrating how proteins can influence rRNA interactions distant from their immediate binding sites, leading to cooperative assembly of the ribosomal subunits. In vivo footprinting of immature ribosomes inside bacterial cells was accomplished using a cryo-sample holder and fast shutter. The results show how defects in ribosome assembly accumulate under stress conditions, when cellular maturation factors that normally assure proper assembly are absent.