Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently

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

Diattenuation of Brain Tissue and its Impact on 3D Polarized Light Imaging

3D-Polarized Light Imaging (3D-PLI) reconstructs nerve fibers in histological brain sections by measuring their birefringence. This study investigates another effect caused by the optical anisotropy of brain tissue - diattenuation. Based on numerical and experimental studies and a complete analytical description of the optical system, the diattenuation was determined to be below 4 % in rat brain tissue. It was demonstrated that the diattenuation effect has negligible impact on the fiber orientations derived by 3D-PLI. The diattenuation signal, however, was found to highlight different anatomical structures that cannot be distinguished with current imaging techniques, which makes Diattenuation Imaging a promising extension to 3D-PLI.Comment: 32 pages, 15 figure

Popculture in postcolonial literature Motifs of popular culture in Arundhati Roy’s The God of Small Things and Eden Robinson’s Monkey Beach

The reaction of LGa (L = Dipp­(4-(Dipp-imino)­pent-2-en-2-yl)­amide; Dipp: 2,6-diisopropylphenyl) and white phosphorus was revisited. A plethora of unprecedented polyphosphanes in addition to the known monoinserted product LGaP₄ (<b>1</b>) are observed. An optimized synthesis of the hitherto unknown hexaphosphane (LGa)₂P₆ (<b>3</b>) is presented, and its subsequent selective derivatization with Brønsted acids, MeOTf, Ph₂ECl (E = P, As), and NaOCP provides access to a wealth of functionalized hexa- and heptaphosphanes

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

Satellitengestützte Vermessung von städtischem Grün in deutschen Städten

Urbane Grünflächen besitzen vielfältige Funktionen und sind als Bereitsteller von Ökosystemleistungen von zentraler Bedeutung. Sie dienen als Naherholungsflächen für die Stadtbevölkerung, als Lebensraum für Flora und Fauna und verbessern die Luftqualität. Mit Daten des europäischen Erdbeobachtungsprogramms Copernicus werden Satellitendaten in einer hohen geometrischen Auflösung sowie mit einer hohen räumlichen und zeitlichen Abdeckung kostenlos zur Verfügung gestellt. Die Aufnahmen der Sentinel-2 Satelliten des Copernicus Programms werden in dieser Analyse verwendet, um urbane Grünflächen in deutschen Städten zu kartieren. Um phänologische Einflüsse abzubilden wird der Jahresgang der Vegetation anhand eines Medianmosaiks bzw. über Vegetationsindizes berücksichtigt. Darauf aufbauend wurde eine Methodik zur Landnutzungs-/Landbedeckungsklassifikation auf Basis von LUCAS Referenzpunkten entwickelt und getestet. Die hohe Gesamtgenauigkeit von 92,3 % zeigt, dass innerstädtische Grünflächen mithilfe flächendeckender Satellitendaten in hohem Detailgrad erfasst werden können

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

EU cohesion policy on the ground: Analyzing small-scale effects using satellite data

We present a novel approach to analyze the effects of EU cohesion policy on local economic activity. For all municipalities in the border area of the Czech Republic, Germany, and Poland, we collect project-level data on EU funding in the period between 2007 and 2013. Using night light emission data as a proxy for economic development, we show that receiving a higher amount of EU funding is associated with increased economic activity at the municipal level. Our paper demonstrates that remote sensing data can provide an effective way to model local economic development also in Europe, where comprehensive cross-border data are not available at such a spatially granular level

A fast and efficient translational control system for conditional expression of yeast genes

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5'UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression

Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer

Publisher's version (útgefin grein)The ability of the cytidine analog Ç m f to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Ç m f-labeled single-and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Ç m f. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Ç m f at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Center (CRC) 902; ‘Molecular Principles of RNA-based Regulation’ sub-projects A7, B14 and Mercator Fellowship. Funding for open access charge: DFG (CRC902); sub-projects A7, B14 and Mercator Fellowship.Peer Reviewe