Visualizing spatially correlated dynamics that directs RNA conformational transitions

RNAs fold into three-dimensional (3D) structures that subsequently undergo large, functionally important, conformational transitions in response to a variety of cellular signals. RNA structures are believed to encode spatially tuned flexibility that can direct transitions along specific conformational pathways. However, this hypothesis has proved difficult to examine directly because atomic movements in complex biomolecules cannot be visualized in 3D by using current experimental methods. Here we report the successful implementation of a strategy using NMR that has allowed us to visualize, with complete 3D rotational sensitivity, the dynamics between two RNA helices that are linked by a functionally important trinucleotide bulge over timescales extending up to milliseconds. The key to our approach is to anchor NMR frames of reference onto each helix and thereby directly measure their dynamics, one relative to the other, using 'relativistic' sets of residual dipolar couplings (RDCs). Using this approach, we uncovered super-large amplitude helix motions that trace out a surprisingly structured and spatially correlated 3D dynamic trajectory. The two helices twist around their individual axes by approximately 53 degrees and 110 degrees in a highly correlated manner (R = 0.97) while simultaneously (R = 0.81-0.92) bending by about 94 degrees. Remarkably, the 3D dynamic trajectory is dotted at various positions by seven distinct ligand-bound conformations of the RNA. Thus even partly unstructured RNAs can undergo structured dynamics that directs ligand-induced transitions along specific predefined conformational pathways.     

Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.     

A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

Lipases are hydrolytic enzymes which break down triacylglycerides into free fatty acids and glycerols. They have been classified as serine hydrolases owing to their inhibition by diethyl p-nitrophenyl phosphate. Lipase activity is greatly increased at the lipid-water interface, a phenomenon known as interfacial activation. X-ray analysis has revealed the atomic structures of two triacylglycerol lipases, unrelated in sequence: the human pancreatic lipase (hPL)4, and an enzyme isolated from the fungus Rhizomucor (formerly Mucor) miehei (RmL). In both enzymes the active centres contain structurally analogous Asp-His-Ser triads (characteristic of serine proteinases), which are buried completely beneath a short helical segment, or 'lid'. Here we present the crystal structure (at 3 A resolution) of a complex of R. miehei lipase with n-hexylphosphonate ethyl ester in which the enzyme's active site is exposed by the movement of the helical lid. This movement also increases the nonpolarity of the surface surrounding the catalytic site. We propose that the structure of the enzyme in this complex is equivalent to the activated state generated by the oil-water interface.     

Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain

RNA interference is a conserved mechanism that regulates gene expression in response to the presence of double-stranded (ds)RNAs. The RNase III-like enzyme Dicer first cleaves dsRNA into 21-23-nucleotide small interfering RNAs (siRNAs). In the effector step, the multimeric RNA-induced silencing complex (RISC) identifies messenger RNAs homologous to the siRNAs and promotes their degradation. The Argonaute 2 protein (Ago2) is a critical component of RISC. Both Argonaute and Dicer family proteins contain a common PAZ domain whose function is unknown. Here we present the three-dimensional nuclear magnetic resonance structure of the Drosophila melanogaster Ago2 PAZ domain. This domain adopts a nucleic-acid-binding fold that is stabilized by conserved hydrophobic residues. The nucleic-acid-binding patch is located in a cleft between the surface of a central beta-barrel and a conserved module comprising strands beta3, beta4 and helix alpha3. Because critical structural residues and the binding surface are conserved, we suggest that PAZ domains in all members of the Argonaute and Dicer families adopt a similar fold with nucleic-acid binding function, and that this plays an important part in gene silencing.     

RNA-guided genetic silencing systems in bacteria and archaea

Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.     

Structure of the E. coli signal recognition particle bound to a translating ribosome

The prokaryotic signal recognition particle (SRP) targets membrane proteins into the inner membrane. It binds translating ribosomes and screens the emerging nascent chain for a hydrophobic signal sequence, such as the transmembrane helix of inner membrane proteins. If such a sequence emerges, the SRP binds tightly, allowing the SRP receptor to lock on. This assembly delivers the ribosome-nascent chain complex to the protein translocation machinery in the membrane. Using cryo-electron microscopy and single-particle reconstruction, we obtained a 16 A structure of the Escherichia coli SRP in complex with a translating E. coli ribosome containing a nascent chain with a transmembrane helix anchor. We also obtained structural information on the SRP bound to an empty E. coli ribosome. The latter might share characteristics with a scanning SRP complex, whereas the former represents the next step: the targeting complex ready for receptor binding. High-resolution structures of the bacterial ribosome and of the bacterial SRP components are available, and their fitting explains our electron microscopic density. The structures reveal the regions that are involved in complex formation, provide insight into the conformation of the SRP on the ribosome and indicate the conformational changes that accompany high-affinity SRP binding to ribosome nascent chain complexes upon recognition of the signal sequence.     

Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion

The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix. Viral fusion proteins consist of a membrane-disturbing 'fusion peptide' and a helical bundle that pin the membranes together. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events: formation of helical bundles by SNAREs precedes hemifusion; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion: both use a docking mechanism via helical bundles and additional peptides to destabilize the membrane and efficiently induce lipid mixing. Our results suggest that a common lipidic intermediate may underlie all fusion reactions of lipid bilayers.     

Spliceosomal RNA U6 is remarkably conserved from yeast to mammals

The small nuclear RNA U6 and its gene have been isolated from yeast. In striking contrast to other yeast spliceosomal RNAs, U6 is very similar in size, sequence and structure to its mammalian homologue. The single-copy gene is essential. These properties suggest a central role in pre-mRNA processing. An extensive base-pairing interaction with U4 snRNA is described; the destabilization of the U4/U6 complex seen during splicing thus requires a large conformational change.     

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB

Accurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome.     

A potential donor gene for the bm1 gene conversion event in the C57BL mouse

The mammalian major histocompatibility complex (MHC; H-2 complex in mouse) is a large multigene complex which encodes cell-surface antigens involved in the cellular immune response to foreign antigens. Class I polypeptides expressed at the H-2K and H-2D loci of numerous mouse strains exhibit an unusually high degree of genetic polymorphism, which is assumed to be related to their function as primary recognition elements in the immune response. We suggested that this H-2 polymorphism may arise by gene conversion-like events between non-allelic class I genes. This is supported by our recent comparison of the DNA sequences of the normal H-2Kb gene sequence, from the C57BL/10 mouse, and a mutant form of this gene called H-2Kbm1: the mutant allele differs from the H-2Kb gene in seven bases out of a region of 13 bases in exon 3 of the class I gene (which encodes alpha 2 (C1) the second highly polymorphic protein domain), suggesting that this region of new sequence had been introduced into the H-2Kb sequence following unequal pairing of two class I genes in the genome of the C57BL mouse. Schulze et al. have obtained similar results. Here we report work identifying a potential donor gene in our library of 26 class I genes cloned from the C57BL/10 mouse.     

The crystal structure of dynamin

Dynamin-related proteins (DRPs) are multi-domain GTPases that function via oligomerization and GTP-dependent conformational changes to play central roles in regulating membrane structure across phylogenetic kingdoms. How DRPs harness self-assembly and GTP-dependent conformational changes to remodel membranes is not understood. Here we present the crystal structure of an assembly-deficient mammalian endocytic DRP, dynamin 1, lacking the proline-rich domain, in its nucleotide-free state. The dynamin 1 monomer is an extended structure with the GTPase domain and bundle signalling element positioned on top of a long helical stalk with the pleckstrin homology domain flexibly attached on its opposing end. Dynamin 1 dimer and higher order dimer multimers form via interfaces located in the stalk. Analysis of these interfaces provides insight into DRP family member specificity and regulation and provides a framework for understanding the biogenesis of higher order DRP structures and the mechanism of DRP-mediated membrane scission events.     

microRNA对肿瘤细胞增殖与分化的调控

microRNA(miRNA)是一种长度约为22核苷酸(nt)的非编码RNA,其主要通过碱基互补与靶mRNA的3'端非翻译区(3'UTR)结合,导致靶mRNA降解或抑制蛋白质的合成,在转录后水平调节基因的表达.miRNA突变、缺失或表达水平的异常会导致生理的异常与疾病的发生,与人类肿瘤疾病密切相关,它具有类似于癌基因或抑癌基因的作用,可参与肿瘤细胞的增殖、分化和细胞凋亡等调控过程.miRNA在肿瘤诊断和治疗方面具有广阔的应用前景.     

An incipient 3(10) helix in Piv-Pro-Pro-Ala-NHMe as a model for peptide folding

The molecular mechanism of helix nucleation in peptides and proteins is not yet understood and the question of whether sharp turns in the polypeptide backbone serve as nuclei for protein folding has evoked controversy. A recent study of the conformation of a tetrapeptide containing the stereochemically constrained residue alpha-aminoisobutyric acid, both in solution and the solid state, yielded a structure consisting of two consecutive beta-turns, leading to an incipient 3(10) helical conformation. :This led us to speculate that specific tri- and tetrapeptide sequences may indeed provide a helical twist to the amino-terminal segment of helical regions in proteins and provide a nucleation site for further propagation. The transformation from a 3(10) helical structure to an alpha-helix should be facile and requires only small changes in the phi and psi conformational angles and a rearrangement of the hydrogen bonding pattern. If such a mechanism is involved then it should be possible to isolate an incipient 3(10) helical conformation in a tripeptide amide or tetrapeptide sequence, based purely on the driving force derived from short-range interactions. We have synthesised and studied the model peptide pivaloyl-Pro-Pro-Ala-NHMe (compound I) and provide here spectroscopic evidence for a 3(10) helical conformation in compound I.     

Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra

DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute molecular computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large numbers (hundreds) of unique DNA strands poses a challenging design problem. Here, we demonstrate a simple solution to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger three-dimensional structures. We test this hierarchical self-assembly concept with DNA molecules that form three-point-star motifs, or tiles. By controlling the flexibility and concentration of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometres in size and comprised of four, twenty or sixty individual tiles, respectively. We expect that our assembly strategy can be adapted to allow the fabrication of a range of relatively complex three-dimensional structures.