title: Use of the U1A Protein to Facilitate Crystallization and Structure Determination of Large RNAs.
abstract: The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described.
authors.affiliation: Laboratory of RNA Biophysics and Cellular Physiology, National Heart, Lung and Blood Institute, 50 South Drive, MSC8012, Bethesda, MD, 20892-8012, USA, adrian.ferre@nih.gov.
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
mesh_terms: D001665:Binding Sites; D003460:Crystallization / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000032:analysis / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry*; D012263:Ribonucleosides / Q000737:chemistry; D017381:Spliceosomes / Q000378:metabolism; D013752:Tetracycline / Q000737:chemistry
chemical_list: D010710:Phosphates; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012263:Ribonucleosides; C075339:U1A protein; D012313:RNA; D013752:Tetracycline