Download
1 / 36
360 likes | 449 Views
The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution. Nenad Ban, 1* Poul Nissen, 1* Jeffrey Hansen, 1 Peter B. Moore, 1, 2 Thomas A. Steitz 1, 2, 3 1 Department of Molecular Biophysics & Biochemistry, 2 Department of Chemistry, Yale University,
E N D
The Complete Atomic Structure of the Large Ribosomal Subunit at 2.4 Å Resolution Nenad Ban,1* Poul Nissen,1* Jeffrey Hansen,1 Peter B. Moore,1, 2 Thomas A. Steitz1, 2, 3 1Department of Molecular Biophysics & Biochemistry, 2 Department of Chemistry, Yale University, 3 Howard Hughes Medical Institute, New Haven, CT 06520-8114, USA.
The large subunit of ribosome in prokaryotes : 23S rRNA+ 5S rRNA +31 proteins The activity : catalyzes peptide bond formation--peptidyltransferase--and the binding site for the G-protein (GTP-bindingprotein) factors that assist in the initiation, elongation, andtermination phases of protein synthesis.
Why to do this research? Structure* Synthesis Mechanism DNA polymerase known DNA clear RNA polymerase known RNA clear 50S subnuit of unknown Protein mystery Ribosome
Gradual progress in demonstrate the structure of the large subunit: At first, 3D electron microscopicimages: visualize many of the proteins and nucleic acids thatassist in protein synthesis bound to the ribosome (3). Then, 9 Å resolution x-ray crystallographicmap: featuresrecognizable as duplex RNA of the large subunit from H.marismortui. 7.8Å resolution map of the entire T.thermophilusribosome: the positions of tRNA molecules bound to itsA, P, and E sites . 7.5 Å resolution electronmicroscopic map (earlier this year) an approximate model of the RNA structure inthe large subunit from E. coli
5.5 Å resolution map ofthe 30S subunit from T. thermophilus :the fitting of solved protein structures and the interpretationof some of its RNA features. 4.5 Å resolution map of the T. thermophilus30S subunit was published. 2.4 Å resolution electron density map: an atomic structure of the H. marismortui50S ribosomal. The model includes 2711 of the 2923 nucleotidesof 23S rRNA, all 122 nucleotides of its 5S rRNA,and structures for the 27 proteins that are well ordered in thesubunit.
This paper describes the architecture of the subunit, thestructure of its RNAs, and discuss the location, structures, andfunctions of its proteins based on the map of 2.4 Å resolution.
I. Structure determination. How to extend the resolution of the map of H. marismortui 50S ribosomal subunit from 5 to 2.4Å: A back-extraction procedure was developed for reproduciblygrowing crystals that are much thicker than those available earlierand that diffract to at least 2.2 Å resolution. The twinning ofcrystalswas eliminated by adjusting crystal stabilization conditions C. Most of the x-ray data used for high-resolution phasing were collectedat the Brookhaven National Synchrotron Light Source. Osmium pentamine(132 sites) and iridium hexamine (84 sites) derivatives were used in in producing isomorphous replacementand anomalous scattering phase information D. With thesolvent-flipping procedure in the CNS program.
II. Sequence fitting and protein identification. The sequence of 23S rRNA was fit into theelectron density map nucleotide by nucleotide starting from itssarcin/ricin loop sequence [A2691 to A2702 ]. The remaining RNA electron density neatly accommodated5S rRNA. ~4000 amino acid residues of 27 proteins werefit into electron density. The structures of proteins determined newly in this study: L3,L5,L7,L13,L15-21,L23-9,L18e,L21e,L24e,L31e,L32e, L37e,L39e,L44e,L7ae,L10e,L15e,L37ae. Totally 27 . Note: Sequences of 28 proteins in Swiss-Prot data bank.
The secondary structure of this 23S rRNA consists of a central loop that is closed by a terminal stem, from which 11 moreor less complicated stem-loops radiate. It is customary to describethe molecule as consisting of six domains and to number its helicalstems sequentially starting from the 5' end.
V.Overall architecture of rRNA The six domains of 23S rRNA and 5S rRNA all have complicated, convoluted shapes thatfit together to produce a compact, monolithic RNA mass. In three dimensions the large subunit is a single,gigantic domain.
The tertiary and secondary structures of the RNA in the H. marismortui large ribosomal subunit and its domains. (A and B)
DI: look likes a mushroom, lies in the back of the particle and behind and below L1. D II: the largest, accounting for most of the back of the particle; D III: a compact globular domain at the bottom left region; D IV: accounts for most of the interface that contacts the 30S subunit; D V: between D IV and II, intimately involved in the peptidyl transferase activity of the ribosome; D VI: the smallest, forms a large part of the surface immediately below the L7/L12 stalk.
VI. Sequence conservation and interactions in 23S rRNAA. There are two classes of conserved sequences in 23S rRNA. One contains residues concentrated in the active site regions ofthe large subunit for substrate binding, factor binding, andcatalytic activity. The second class consists of much shorter sequencesscattered throughout the particle (Fig. 5, red sequences), they are involved in the inter- and intradomaininteractions that stabilize the tertiary structure of 23S rRNA.B.Thepredominance of adenosines among the conserved residues in rRNAsis useful to help tertiary interactions.
Kind of interactions in 50S subunit: Ribose zipper and the tetraloop-tetraloopreceptor interaction A-dependent motifs interraction RNA-protein interaction 5S rRNA and 23S rRNA interactions Backbone-backbone interactions etc.
VII. Proteins. The structures of 27 proteins in the large ribosomal subunit of H. marismortui (Table 2) have been determined in this study. 21 of these protein structures have not been previouslyestablished for any homologs. The structures of the six thatdo have homologs of known structure have been rebuilt into theelectron density map with their H. marismortui sequences. There are structures available for homologs of H. marismortuiL1, L11, and L12, which cannot be visualized in the 2.4 Å resolutionelectron density map. Only the structure of L10 is still unknownamong the 31 proteins of this subunit.
Of the 30 large subunit ribosomal proteins, 17 are globular proteins. The remaining 13 proteins either have globular bodies with extensionsprotruding from them ("glb+ext") or are entirely extended ("ext").Their extensions often lack obvious tertiary structure and inmany regions are devoid of significant secondary structure aswell (Fig. 6).
Most proteins of the 50S subunitdo not extend significantly beyond the envelope defined by theRNA (Fig. 7). Their globular domains are found largelyon the particle's exterior, often nestled in the gaps and crevicesformed by the folding of the RNA. The proteins of the large ribosomal subunitdo not form a shell around the nucleic acid with which they associate.The proteins do not become surroundedby nucleic acid. Instead, the proteins act like mortarfilling the gaps and cracks between "RNA bricks."
蛋白质延伸入RNA和 多域的相互作用
There are only a few segmentsof the 23S rRNA that do not interact with protein at all Of the2923 nucleotides in 23S rRNA, 1157 make at least van der Waalscontact with protein (Fig. 8D), and there are only 10sequences longer than 20 nucleotides in which no nucleotide contactsprotein. The longest such sequence contains 47 nucleotides, andis the part of domain IV that forms the ridge of the active sitecleft. All of the proteins in the particle except L12 interact directly with RNA, among the 30 proteins, 23 interactwith two rRNA domains or more (Table 2). L22 interactswith RNA sequences belonging to all six domains of the 23S rRNA.
Every rRNAdomain interacts with multiple proteins. DomainV interacts with 15 proteins, some intimately anda few in passing. Of the seven proteins that interact with only one domain, three (L1, L10, and L11) participate directly in the protein synthesisprocess. Another three (L24, L29, and L18e) interactwith several secondary structure elements within the domains towhich they bind, and presumably they function to stabilize thetertiary structures of their domains. The last of the single RNAdomain proteins, L7ae, is puzzling. It cannot function as an RNAstabilizing protein because it interacts with only a single sequencein domain I. It could also be involved inthe 70S assembly, because L7ae was originally assigned as a smallsubunit protein (HMS6). L1 appearsto be important for E-site function (50), and maybeit is involved in that activity.
While many ribosomal proteins interact primarily with RNA, a few interact significantly with other proteins. The most strikingstructure generated by protein-protein interactions is the proteincluster composed of L3, L6, L13, L14, and L24e that is found closeto the factor binding site. The surface of these proteins providesimportant interactions with factors. It may prove to be more generallythe case that ribosomal proteins interacting primarily with RNAare principally stabilizing RNA structure, whereas some of thoseshowing extensive protein-protein interactions may have additionalbinding functions
Weakness of the approach adopted in this study: For obvious reasons, the structures of the extended tails andloops of ribosomal proteins cannot be determined in the absenceof the RNAs that give them structure, and the feasibility of strategiesthat depend on producing low-molecular weight RNA-protein complexesthat have all the RNA contacts required to fix the structuresof such proteins seems remote.
Still to demonstrate in future: A. The principles of protein-RNA interaction thatshould emerge from the 27 protein complexes with RNA have yetto be developed. B. Most of the important RNA secondaryand tertiary structural motifs are to be found in nature . C. It will be interesting to see whether a complete analysis of thisRNA structural database will enable the prediction of structuresfor other RNA sequences D. Enormous numbers ofmonovalent and divalent metal ions as well as water moleculesare visible in this map. Analysis of their interactions with RNAshould elucidate their roles in the formation and stabilizationof RNA structure.
