Building a map of the dynamic ribosome

Abstract

Our understanding of the static structure of the 80S eukaryotic ribosome has been enhanced by the emergence of high resolution cryo-electron microscopy and crystallography data over the past 15 years. However our understanding of the dynamic nature of the ribosome has lagged. High-throughput Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (hSHAPE) is easily amenable for interrogation of rRNA dynamics. Here we report an improved method of hSHAPE data analysis and apply it to translation initiation and elongation complexes of the yeast ribosome to identify the changes in rRNA flexibility that occur during these processes. Most importantly, we have obtained complete analyses of tRNA binding and intersubunit bridge dynamics, as well as overall expansion segment dynamics, as the ribosome progresses through the translation elongation cycle. The results from these analyses suggest that (1) the yeast P site tRNA binding site is a "hybrid" between the prokaryotic and mammalian P sites, (2) there may be substates of intersubunit rotation, (3) expansion segments may have roles in accommodation. We are also able to identify a network of information pathways that connect elongation factor binding sites to all tRNA binding sites, five intersubunit bridges and two expansion segments. Future directions of this project will focus on improving the visualization of our data to better reflect the highly dynamic nature of the yeast ribosome and to reveal the underlying causes of the observed rRNA flexibility changes.