Essential role of cyclization sequences in Flavivirus RNA replication

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first similar to 160 nucleotides) and the 3' untranslated region (last similar to 115 nucleotides) for a range of mosquito borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus,were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

Collective dynamic properties in simple crystals: Influence of the structural disorder

Collective dynamic properties in Lennard-Jones crystals are investigated by molecular dynamics simulation. The study is focused on properties such as the dynamic structure factors, the longitudinal and transverse currents and the density of states. The influence on these properties of the structural disorder is analyzed by comparing the results for one-component crystals with those for liquids and supercooled liquids at analogous conditions. The effects of species-disorder on the collective properties of binary crystals are also discussed.

Dynamic properties of Lennard Jones fluids and liquid metals

The dependence of the dynamic properties of liquid metals and Lennard-Jones fluids on the characteristics of the interaction potentials is analyzed. Molecular-dynamics simulations of liquids in analogous conditions but assuming that their particles interact either through a Lennard-Jones or a liquid-metal potential were carried out. The Lennard-Jones potentials were chosen so that both the effective size of the particles and the depth of the potential well were very close to those of the liquid-metal potentials. In order to investigate the extent to which the dynamic properties of liquids depend on the short-range attractive interactions as well as on the softness of the potential cores, molecular-dynamics simulations of the same systems but assuming purely repulsive interactions with the same potential cores were also performed. The study includes both singleparticle dynamic properties, such as the velocity autocorrelation functions, and collective dynamic properties, such as the intermediate scattering funcfunctions, and collective dynamic properties, such as the intermediate scattering functions, the dynamic structure factors, the longitudinal and transverse current correlations, and the transport coefficients.

Collective dynamic properties in simple crystals: Influence of the structural disorder

Collective dynamic properties in Lennard-Jones crystals are investigated by molecular dynamics simulation. The study is focused on properties such as the dynamic structure factors, the longitudinal and transverse currents and the density of states. The influence on these properties of the structural disorder is analyzed by comparing the results for one-component crystals with those for liquids and supercooled liquids at analogous conditions. The effects of species-disorder on the collective properties of binary crystals are also discussed.

Water-in-Oil Micro-Emulsion Enhances the Secondary Structure of a Protein by Confinement

A scheme is presented in which an organic solvent environment in combination with surfactants is used to confine a natively unfolded protein inside an inverse microemulsion droplet. This type of confinement allows a study that provides unique insight into the dynamic structure of an unfolded, flexible protein which is still solvated and thus under near-physiological conditions. In a model system, the protein osteopontin (OPN) is used. It is a highly phosphorylated glycoprotein that is expressed in a wide range of cells and tissues for which limited structural analysis exists due to the high degree of flexibility and large number of post-translational modifications. OPN is implicated in tissue functions, such as inflammation and mineralisation. It also has a key function in tumour metastasis and progression. Circular dichroism measurements show that confinement enhances the secondary structural features of the protein. Small-angle X-ray scattering and dynamic light scattering show that OPN changes from being a flexible protein in aqueous solution to adopting a less flexible and more compact structure inside the microemulsion droplets. This novel approach for confining proteins while they are still hydrated may aid in studying the structure of a wide range of natively unfolded proteins.

Influence of chain stiffness on structure and dynamics of polymers in the melt

Die vorliegende Arbeit beschaeftigt sich mit der Untersuchung vonPolymeren mit intrinsischer Steifigkeit. Es werden vor allem lokale statische unddynamische Eigenschaften anhand zweier verschiedener Simulationsmodellebetrachtet: Ein generisches Polymermodell, bei dem nur dieSteifigkeit als ein das spezifische Polymer charakterisierenden Parametereingeht und ein atomistisches Modell fuer trans-Polyisopren. Mit Hilfe des ersten Modells koennen Statik und Dynamik wurmartiger Kettenbeobachtet werden. Das Blob-Konzept ist eine angemessene statischeBeschreibung. Lokale Orientierungen haengen schwach von derSteifigkeit ab. Das Reptationsmodell kann die beobachtete Dynamik fuer lange Kettennicht mehr angemessen beschreiben. Lange Ketten bewegen sich, als obsie in Roehren gezwaengt waeren; jedoch ist die Bewegung starkabhaengig von der Steifigkeit. Fuer Ketten dieser Art konntequalitativ das Verhalten reproduziert werden, das in NMR-Experimentenbeobachtet wird. Eine Verhakungslaenge laesst sich fuer solche Kettenkaum mehr definieren. Dynamische Strukturfunktionen und insbesonderedie direkte Visualisierung der Ketten verdeutlichen die effektiv aufeine Roehre beschraenkte Bewegung. Das atomistische Polyisoprenmodell wurde mit verschiedenen Experimenten,verglichen. In den Simulationen bei konnten qualitativ undsemiquantitativ experimentelle Ergebnisse reproduziert werden. Zuletzt wurden die Laengen- und Zeitskalen der beiden Modelleerfolgreich aufeinander abgebildet.

Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure

Single chicken erythrocyte chromatin fibers were stretched and released at room temperature with force-measuring laser tweezers. In low ionic strength, the stretch-release curves reveal a process of continuous deformation with little or no internucleosomal attraction. A persistence length of 30 nm and a stretch modulus of ≈5 pN is determined for the fibers. At forces of 20 pN and higher, the fibers are modified irreversibly, probably through the mechanical removal of the histone cores from native chromatin. In 40–150 mM NaCl, a distinctive condensation-decondensation transition appears between 5 and 6 pN, corresponding to an internucleosomal attraction energy of ≈2.0 kcal/mol per nucleosome. Thus, in physiological ionic strength the fibers possess a dynamic structure in which the fiber locally interconverting between “open” and “closed” states because of thermal fluctuations.

Conserved stem–loop structures in the HIV-1 RNA region containing the A3 3′ splice site and its cis-regulatory element: possible involvement in RNA splicing

The HIV-1 transcript is alternatively spliced to over 30 different mRNAs. Whether RNA secondary structure can influence HIV-1 RNA alternative splicing has not previously been examined. Here we have determined the secondary structure of the HIV-1/BRU RNA segment, containing the alternative A3, A4a, A4b, A4c and A5 3′ splice sites. Site A3, required for tat mRNA production, is contained in the terminal loop of a stem–loop structure (SLS2), which is highly conserved in HIV-1 and related SIVcpz strains. The exon splicing silencer (ESS2) acting on site A3 is located in a long irregular stem–loop structure (SLS3). Two SLS3 domains were protected by nuclear components under splicing condition assays. One contains the A4c branch points and a putative SR protein binding site. The other one is adjacent to ESS2. Unexpectedly, only the 3′ A residue of ESS2 was protected. The suboptimal A3 polypyrimidine tract (PPT) is base paired. Using site-directed mutagenesis and transfection of a mini-HIV-1 cDNA into HeLa cells, we found that, in a wild-type PPT context, a mutation of the A3 downstream sequence that reinforced SLS2 stability decreased site A3 utilization. This was not the case with an optimized PPT. Hence, sequence and secondary structure of the PPT may cooperate in limiting site A3 utilization.

Smoothness within ruggedness: the role of neutrality in adaptation.

RNA secondary structure folding algorithms predict the existence of connected networks of RNA sequences with identical structure. On such networks, evolving populations split into subpopulations, which diffuse independently in sequence space. This demands a distinction between two mutation thresholds: one at which genotypic information is lost and one at which phenotypic information is lost. In between, diffusion enables the search of vast areas in genotype space while still preserving the dominant phenotype. By this dynamic the success of phenotypic adaptation becomes much less sensitive to the initial conditions in genotype space.

Preliminary crystallographic characterization of an RNA helicase from Kunjin virus

Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3' nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds.

Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

Defects in vesicle core induced by Escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 1 0-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.

Bayesian ratemaking procedure of crop insurance contracts with skewed distribution

Over the years, crop insurance programs became the focus of agricultural policy in the USA, Spain, Mexico, and more recently in Brazil. Given the increasing interest in insurance, accurate calculation of the premium rate is of great importance. We address the crop-yield distribution issue and its implications in pricing an insurance contract considering the dynamic structure of the data and incorporating the spatial correlation in the Hierarchical Bayesian framework. Results show that empirical (insurers) rates are higher in low risk areas and lower in high risk areas. Such methodological improvement is primarily important in situations of limited data.

Experimentation with accumulation

We study signal-dependent experimentation in the presence of accumulation and show that the passive-learner’s action surprisingly coincides with the experimentor’s when the unknown term is the one determining the decay rate of the stock, while they differ when the parameter being learned is the one measuring the accumulation rate. These results highlight the importance of the dynamic structure of the problem in signal-dependent experimentation. Moreover, they have important consequences for the pollution-accumulation debate currently in progress.

High-Momentum Response of Liquid 3He

A final-state-effects formalism suitable to analyze the high-momentum response of Fermi liquids is presented and used to study the dynamic structure function of liquid 3He. The theory, developed as a natural extension of the Gersch-Rodriguez formalism, incorporates the Fermi statistics explicitly through a new additive term which depends on the semidiagonal two-body density matrix. The use of a realistic momentum distribution, calculated using the diffusion Monte Carlo method, and the inclusion of this additive correction allows for good agreement with available deep-inelastic neutron scattering data.