Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.     

Crystal structure of an initiation factor bound to the 30S ribosomal subunit

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.     

Crystal structure of the eukaryotic ribosome

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.     

Distribution of protein and RNA in the 30S ribosomal subunit

In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms.     

Crystal structure of the RC-LH1 core complex from Rhodopseudomonas palustris

The crystal structure at 4.8 angstrom resolution of the reaction center-light harvesting 1 (RC-LH1) core complex from Rhodopseudomonas palustris shows the reaction center surrounded by an oval LH1 complex that consists of 15 pairs of transmembrane helical alpha- and beta-apoproteins and their coordinated bacteriochlorophylls. Complete closure of the RC by the LH1 is prevented by a single transmembrane helix, out of register with the array of inner LH1 alpha-apoproteins. This break, located next to the binding site in the reaction center for the secondary electron acceptor ubiquinone (UQB), may provide a portal through which UQB can transfer electrons to cytochrome b/c1.     

Recognition of cognate transfer RNA by the 30S ribosomal subunit

Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or "wobble," position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs.     

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.     

Crystal structure of the 1: 1 complex of 5-fluorouracil and 9-ethylhypoxanthine

9-Ethylhypoxanthine and 5-fluorouracil form a 1 : 1 crystalline complex. The structure of this complex has been solved by x-ray diffraction analysis. The molecules crystallize in a monoclinic lattice and form a sheet structure in which pairs of fluorouracil molecules are held together by two hydrogen bonds. The 9-ethylhypoxanthine residues fill up the rest of the molecular sheet by forming single hydrogen bonds with each uracil pair.     

Secondary structure of 16S ribosomal RNA

A secondary structure model for 16S ribosomal RNA which is based on available chemical, enzymatic, and comparative sequence data shows good agreement between constraints dictated by the model and a wide variety of experimental observations. The four major structural domains created by the base-pairing scheme correspond closely to RNA fragments isolated after nuclease digestion in the presence of bound ribosomal proteins. Functionally important sites appear to be located in unpaired regions and are phylogenetically highly conserved.     

Crystal structure of the rabies virus nucleoprotein-RNA complex

Negative-strand RNA viruses condense their genome into a helical nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies virus nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by nucleoproteins is likely a common mechanism used by negative-strand RNA viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.     

The complete atomic structure of the large ribosomal subunit at 2.4 A resolution

The large ribosomal subunit catalyzes peptide bond formation and binds initiation, termination, and elongation factors. We have determined the crystal structure of the large ribosomal subunit from Haloarcula marismortui at 2.4 angstrom resolution, and it includes 2833 of the subunit's 3045 nucleotides and 27 of its 31 proteins. The domains of its RNAs all have irregular shapes and fit together in the ribosome like the pieces of a three-dimensional jigsaw puzzle to form a large, monolithic structure. Proteins are abundant everywhere on its surface except in the active site where peptide bond formation occurs and where it contacts the small subunit. Most of the proteins stabilize the structure by interacting with several RNA domains, often using idiosyncratically folded extensions that reach into the subunit's interior.     

真核细胞翻译起始因子5A(eIF5A)研究进展

真核细胞翻译起始因子5A(eIF5A)是真菌、动植物体内普遍存在的蛋白质翻译起始因子。研究发现其不仅仅在部分蛋白质翻译起始中发挥作用,还在人体癌症发生、促进动植物细胞增殖、细胞衰老和死亡以及一些植物环境胁迫应答等方面都有一定的调控作用。进一步研究真核细胞翻译起始因子5A(eIF5A)的功能,对其生产实践中应用具有非常重要的意义。     

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.     

Crystal structure of the GpIbalpha-thrombin complex essential for platelet aggregation

Direct interaction between platelet receptor glycoprotein Ibalpha (GpIbalpha) and thrombin is required for platelet aggregation and activation at sites of vascular injury. Abnormal GpIbalpha-thrombin binding is associated with many pathological conditions,including occlusive arterial thrombosis and bleeding disorders. The crystal structure of the GpIbalpha-thrombin complex at 2.6 angstrom resolution reveals simultaneous interactions of GpIbalpha with exosite I of one thrombin molecule,and with exosite II of a second thrombin molecule. In the crystal lattice,the periodic arrangement of GpIbalpha-thrombin complexes mirrors a scaffold that could serve as a driving force for tight platelet adhesion. The details of these interactions reconcile GpIbalpha-thrombin binding modes that are presently controversial,highlighting two distinct interfaces that are potential targets for development of novel antithrombotic drugs.     

Crystal structure of a shark single-domain antibody V region in complex with lysozyme

Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies.     

Crystal structure of a complex of acridine, cytosine and water (1:1:1)

X-ray structural analysis of a crystalline complex between acridine C(13)H(9)N, cytosine C(4)H(5)N(3)O, and water (1:1:1) has been completed. The cytosine and water molecules form a sheet-like structure through a series of hydrogen bonds. The acridine molecules are bound to this layer through a hydrogen bridge from the water to the ring nitrogen. The acridine molecules stack in a parallel fashion normal to the cytosine-water sheets, with an average interplanar spacing of 3.5 angstroms.     

植物延伸因子eEF1A研究进展

60年代初,首先从E．coli细胞中分离获得延伸因子,延伸因子eEF1A广泛存在于真核细胞内,是在核糖体上催化氨基酸链的延伸而推动、控制蛋白质的合成等方面起到重要作用的蛋白质因子。在植物蛋白质合成延伸过程中,eEF1A是一个主要的翻译因子;在快速增殖的细胞中,eEF1A基因的表达调控十分保守,其表达水平同细胞生长及增殖速度有关。eEF1A除了参与同翻译控制有关的信号传导外,还参与细胞生长、应激反应及与运动性有关的信号传导,并且与细胞凋亡等有关。eEF1A在体内和体外均能同肌动蛋白纤维及微管蛋白结合,是细胞骨架运动性的调节蛋白。目前许多植物的eEF1A基因已被分离,植物种间的eEF1A氨基酸序列高度保守;植物eEF1A由多基因编码,它的表达受激素、环境胁迫和生长发育过程等因素诱导。文章通过总结植物延伸因子eEF1A的生理作用以及eEF1A基因的克隆、鉴定、诱导表达等分子生物学研究,以期为今后进一步深入研究eEF1A奠定基础。     

Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue

The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.