title: 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 (hSHAPE) 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 SHAPEFinder. 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 trans-acting factors, due to mutations of intrinsic ribosome components or extrinsic factors affecting ribosome biogenesis, or in the presence of translational inhibitors.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Leshin
authors.forename: Ryan
authors.initials: R
authors.lastname: Heselpoth
authors.forename: Ashton Trey
authors.initials: AT
authors.lastname: Belew
authors.forename: Jonathan
authors.initials: J
authors.lastname: Dinman
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D020285:Cryoelectron Microscopy; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012331:RNA, Fungal / Q000737:chemistry*; D012335:RNA, Ribosomal / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D012331:RNA, Fungal; D012335:RNA, Ribosomal