Time-resolved analysis of ribosomal RNA dynamics

Within the last six years the study of the ribosome has fundamentally shifted. There are now atomic and near atomic resolution models from crystallographic studies of the subunits and the 70S ribosome. There are also many structure/function insights from complexes of ligands and the ribosome from medium resolution cryo-EM models. This wealth of information has provided insight into the existence and relevance of several fine and gross conformational changes on the ribosome as it undertakes particular translational tasks. It is now known for certain that the ribosome is a ribozyme and that RNA actively participates in the act of translation. These data will continue to add mechanistic detail to our understanding, but their most promising application is in the generation of testable hypotheses for other biochemical techniques. There is now a structure upon which researchers can hang their collective hats. And with this, blanks between the static structures as well as their functional relevance can be investigated.; Toward this end we have developed a technique that blends the tried and true power of chemical modification structure probing with quench flow fast mixing techniques to provide time resolution of conformational changes on the ribosome as they occur in response to functional ligands. In doing so, the initial studies have shown that the ribosomal subunits form interactions with each other in a stepwise manner suggesting a conformational intermediate in the process. Other data have also shown that mRNA alters the structure of the 30S subunit toward a structure found in the 70S subunit, eliminating the necessity for a post-association structural change. Finally, there are indications that the association of a natural messenger with the 30S subunit through its Shine-Dalgarno sequence may induce a structure that precludes interaction of the mRNA in the A site of the subunit.