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Biochemical Society Transactions (1999) 27 19 The ThreeDimensional Structure o f Bacterial R i b w m a l F W at 13 I! Resolution Richard Brimacanbe, Wax-Plandc-Institut fur Molekulare Genetik, Ihnestrasse 73, 14195 Berlin, Germany. Recent advances i n the cryo-electron microscopy of Escherichia coli ribosomes combined w i t h image processing techniques have led to computerized reconstructions of these ribosomes a t ever higher resolution. A reconstruction of 705 ribosomes carrying t R N A a t the P s i t e and an EF-Tu/tRNA ternary complex stalled w i t h the antibiotic kirromycin at the “pre-A s i t e “ has now been refined to 13 fl i n the laboratories of Or. W. Wintermeyer (Witten, FRG) and Or. M . van Heel (Imperial College, London). A t this resolution, the bound ligands ( t R N A s and EF-Tu) are directly visible i n their entirety, and furthermore many fine structural elements can be seen i n the electron density maps which correspond to individual helices of the rRNA molecules. Previously, models for the 30 folding of the E. coli rRNA molecules have been constructed on the basis of the large body of biochemical data (from cross-linking, foot-printing or similar studies) that has been accumulated over the years. Now, these models can be combined w i t h the EM density maps to derive the 30 structure of the rRNR i n situ w i t h i n the ribosome. W i t h the help of a number of new biochemical data sets, including cross-links between the rRNA and mRNA, t R N A and the growing peptide chain, or w i t h i n and between the 55, 165 and 235 rRNA molecules themselves (collected i n collaboration w i t h the laboratory of Or. 0. Dontsova, Moscow), we have now fitted a l l three rRNA molecules to the 13 1 EM reconstruction mentioned above. The structure produced satisfies both the EM density data and the great majority of the biochemical facts. The X-ray crystallographic structures of t R N A , EF-Tu, and a number of individual ribosomal proteins ( i n collaboration w i t h Dr. 5. White, Memphis USA, Or. V . Ramakrishnan, Salt Lake C i t y USA, Dr. I. Tanaka, Sapporo Japan and Dr. M. Gorlach, Jena FRG) have furthermore been f i t t e d to the rRNA models i n the 705 ribosome, again i n such a way as to satisfy both the biochemical and EM dens i t y constraints. The results are giving u s an increasingly accurate and detailed picture of the structure of the ribosome and of its interactions w i t h functional ligands. I10 RNA Crystallography without RNA Crystalls: Translational Operators, Aptameters and other Motifs P.Stocklev Department of Biology, Uni of Leeds, Leeds LS29JT 111 A89 Molecular interactions in assembly of RNA-binding attenuation protein with RN 2 A.A. Antson, E.J. Dodson & G.G. Dodson, York Structural Biology Laboratory, University of York, York YO1 5DD, UK. X.-P. Chen & P. Gollnick, Department of Biological Sciences, Cooke Hall State University of New York at Buffalo, Buffalo, NY 14260, USA. The X-ray structure of trp RNA-binding attenuation protein (TRAP) from B.stearothermophilus in complex with cognate 53-base (GAGAU),,GAG RNA provides the structural basis for the transcription regulation of trp (trpEDCFBA) operon. In several Bacilli, TRAP regulates transcription of the trp operon in response to changes in the intracellular concentration of L-tryptophan [ 11. TRAP itself is an assembly of 1 1 subunits related by rotational symmetry[2]. When TRAP becomes activated by L-tryptophan it binds specifically to a leader region of the RNA transcript which contains eleven GAG or UAG triplets[3]. By binding to this RNA, TRAP disrupts the formation of a hairpin structure (antiterminator) and allows formation of an alternative, transcription termination hairpin. In the structure, each GAG triplet is bound in a pocket formed by pstrands. The circular arrangement of TRAP subunits generates a belt of 11 GAG binding sites with a diameter of about 80 A. Each RNA triplet is bound specifically with almost no direct interactions formed between the sugar-phosphate backbone and the protein. 1 Gollnick P (1994) Regulation of the Bacillus subtilis tr operon by an RNA-binding protein Molecular Microbiology 11,&I-997. 2 Antson AA, Otrid e J, Brzozowski AM, Dodson El, Dodson GG, Wilson KS, S m i k TM,Yang M, Kurecki T, Gollnick P The structure of trp RNA-binding attenuation protein. Nature 1995, 374: 693-700. 3 Babitzke P, Stults JT, Shire SJ, Yanofsky C (1994) TRAP, the rrp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to reco nise G/UAG repeats in the trpEDCFBA and trpC transcripts. J . dol.Chem. 269: 1659716604. 112 RNA-protein interactions in nuclear pre-mRNA splicing. L. Verdone,A.E. Mayes, I. Dix and J.D. Beggs Institute of Cell and Molecular Biology, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh, EH9 3JR, UK.