Subject(s)
Ribosomes/chemistry , Ribosomes/metabolism , Crystallography, X-Ray , Haloarcula marismortui/enzymology , Haloarcula marismortui/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/genetics , Ribosomal Proteins/chemistry , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsABSTRACT
The 50S subunit of the ribosome catalyzes the peptidyl-transferase reaction of protein synthesis. We have generated X-ray crystallographic electron density maps of the large ribosomal subunit from Haloarcula marismortui at various resolutions up to 9 A using data from crystals that diffract to 3 A. Positioning a 20 A resolution EM image of these particles in the crystal lattice produced phases accurate enough to locate the bound heavy atoms in three derivatives using difference Fourier maps, thus demonstrating the correctness of the EM model and its placement in the unit cell. At 20 A resolution, the X-ray map is similar to the EM map; however, at 9 A it reveals long, continuous, but branched features whose shape, diameter, and right-handed twist are consistent with segments of double-helical RNA that crisscross the subunit.
Subject(s)
Crystallography, X-Ray/methods , Haloarcula marismortui/chemistry , Image Processing, Computer-Assisted/methods , Ribosomes/chemistry , Microscopy, Electron/methods , RNA, Archaeal/chemistry , RNA, Ribosomal, 5S/chemistry , Ribosomal Proteins/chemistryABSTRACT
Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH3)3]2+ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.
Subject(s)
Crystallography, X-Ray/methods , RNA, Ribosomal, 5S/chemistry , Thionucleotides/chemistry , Base Sequence , Bromine , Crystallization , Mercury , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , PlatinumABSTRACT
Two new RNA structures portray how non-Watson-Crick base pairs and metal ions can produce a unique RNA shape suitable for recognition by proteins. The crystal structures of a 62 nt domain of E. coli 5S ribosomal RNA and a duplex dodecamer encompassing an internal loop E have been determined at 3.0 and 1.5 A, respectively. This loop E region is distorted by three "cross-strand purine stacks" and three novel, water-mediated noncanonical base pairs and stabilized by a four metal ion zipper. These features give its minor groove a unique hydrogen-bonding surface and make the adjacent major groove wide enough to permit recognition by the ribosomal protein L25, which is expected to bind to this surface.
Subject(s)
Metals/chemistry , RNA, Ribosomal, 5S/chemistry , Cross-Linking Reagents , Crystallography , Escherichia coli/chemistry , Ions , Nucleic Acid Conformation , Purines/chemistrySubject(s)
RNA, Ribosomal, 5S/isolation & purification , RNA, Ribosomal/isolation & purification , Chromatography, Gel/methods , Crystallography , Escherichia coli/ultrastructure , Hydrogen , Indicators and Reagents , Isotope Labeling/methods , Magnetic Resonance Spectroscopy/methods , Nitrogen Isotopes , RNA, Ribosomal, 5S/ultrastructureABSTRACT
The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.
