Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast

Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression

Numerical Analysis of a New Mixed Formulation for Eigenvalue Convection-Diffusion Problems

A mixed formulation is proposed and analyzed mathematically for coupled convection-diffusion in heterogeneous medias. Transfer in solid parts driven by pure diffusion is coupled with convection-diffusion transfer in fluid parts. This study is carried out for translation-invariant geometries (general infinite cylinders) and unidirectional flows. This formulation brings to the fore a new convection-diffusion operator, the properties of which are mathematically studied: its symmetry is first shown using a suitable scalar product. It is proved to be self-adjoint with compact resolvent on a simple Hilbert space. Its spectrum is characterized as being composed of a double set of eigenvalues: one converging towards −∞ and the other towards +∞, thus resulting in a nonsectorial operator. The decomposition of the convection-diffusion problem into a generalized eigenvalue problem permits the reduction of the original three-dimensional problem into a two-dimensional one. Despite the operator being nonsectorial, a complete solution on the infinite cylinder, associated to a step change of the wall temperature at the origin, is exhibited with the help of the operator’s two sets of eigenvalues/eigenfunctions. On the computational point of view, a mixed variational formulation is naturally associated to the eigenvalue problem. Numerical illustrations are provided for axisymmetrical situations, the convergence of which is found to be consistent with the numerical discretization

Company matters:The presence of other genotypes alters traits and intraspecific selection in an Arctic diatom under climate change

Arctic phytoplankton and their response to future conditions shape one of the most rapidly changing ecosystems on the planet. We tested how much the phenotypic responses of strains from an Arctic diatom population diverge and whether the physiology and intraspecific composition of multi-strain populations differ from expectations based on single strain traits. To this end, we conducted incubation experiments with the diatom Thalassiosira hyalina under present-day and future temperature and pCO2 treatments. Six fresh isolates from the same Svalbard population were incubated as mono- and multi-strain cultures. For the first time, we were able to closely follow intraspecific selection within an artificial population using microsatellites and allele-specific quantitative PCR. Our results show not only that there is substantial variation in how strains of the same species cope with the tested environments, but also that changes in genotype composition, production rates and cellular quotas in the multi-strain cultures are not predictable from monoculture performance. Nevertheless, the physiological responses as well as strain composition of the artificial populations were highly reproducible within each environment. Interestingly, we only detected significant strain sorting in those populations exposed to the future treatment. This study illustrates that the genetic composition of populations can change on very short timescales through selection from the intraspecific standing stock, indicating the potential for rapid population level adaptation to climate change. We further show that individuals adjust their phenotype not only in response to their physico-chemical, but also to their biological surroundings. Such intraspecific interactions need to be understood in order to realistically predict ecosystem responses to global change

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch

Conformational dynamics of the tetracycline-binding aptamer

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer

Chiral symmetry breaking of magnetic vortices by sample roughness

Finite-element micromagnetic simulations are employed to study the chiral symmetry breaking of magnetic vortices, caused by the surface roughness of thin-film magnetic structures. An asymmetry between vortices with different core polarizations has been experimentally observed for square-shaped platelets. E.g., the threshold fields for vortex core switching were found to differ for core up and down. This asymmetry was however not expected for these symmetrically-shaped structures, where both core polarizations should behave symmetrically. Three-dimensional finite element simulations are employed to show that a small surface roughness can break the symmetry between vortex cores pointing up and down. A relatively small sample roughness is found sufficient to reproduce the experimentally observed asymmetries. It arises from the lack of mirror-symmetry of the rough thin-film structures, which causes vortices with different handedness to exhibit asymmetric dynamics

G-flux and Spectral Divisors

We propose a construction of G-flux in singular elliptic Calabi-Yau fourfold compactifications of F-theory, which in the local limit allow a spectral cover description. The main tool of construction is the so-called spectral divisor in the resolved Calabi-Yau geometry, which in the local limit reduces to the Higgs bundle spectral cover. We exemplify the workings of this in the case of an E_6 singularity by constructing the resolved geometry, the spectral divisor and in the local limit, the spectral cover. The G-flux constructed with the spectral divisor is shown to be equivalent to the direct construction from suitably quantized linear combinations of holomorphic surfaces in the resolved geometry, and in the local limit reduces to the spectral cover flux.Comment: 30 page

On Flux Quantization in F-Theory

We study the problem of four-form flux quantization in F-theory compactifications. We prove that for smooth, elliptically fibered Calabi-Yau fourfolds with a Weierstrass representation, the flux is always integrally quantized. This implies that any possible half-integral quantization effects must come from 7-branes, i.e. from singularities of the fourfold. We subsequently analyze the quantization rule on explicit fourfolds with Sp(N) singularities, and connect our findings via Sen's limit to IIB string theory. Via direct computations we find that the four-form is half-integrally quantized whenever the corresponding 7-brane stacks wrap non-spin complex surfaces, in accordance with the perturbative Freed-Witten anomaly. Our calculations on the fourfolds are done via toric techniques, whereas in IIB we rely on Sen's tachyon condensation picture to treat bound states of branes. Finally, we give general formulae for the curvature- and flux-induced D3 tadpoles for general fourfolds with Sp(N) singularities.Comment: 46 page