High throughput structural analysis of yeast ribosomes using hSHAPE

Abstract

Global mapping of rRNA structure by traditional methods is prohibitive in terms of time, labor and expense. High throughput selective 2′ hydroxyl acylation analyzed by primer extension (hSH APE ) bypasses these problems by using fluorescently labeled primers to perform primer extension reactions, the products of which can be separated by capillary electrophoresis, thus enabling long read lengths in a cost effective manner. The data so generated is analyzed in a quantitative fashion using SH APE Finder. This approach was used to map the flexibility of nearly the entire sequences of the 3 largest rRNAs from intact, empty yeast ribosomes. Mapping of these data onto near-atomic resolution yeast ribosome structures revealed the binding sites of known trans-acting factors, as well as previously unknown highly flexible regions of yeast rRNA. Refinement of this technology will enable nucleotide-specific mapping of changes in rRNA structure depending on the status of tRNA occupancy, the presence or absence of other transacting factors, due to mutations of intrinsic ribosome components or extrinsic factors affecting ribosome biogenesis or in the presence of translational inhibitors. © 2011 Landes Bioscience.