Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed ∼200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn. © 2007 Wiley Periodicals, Inc. Biopolymers 85: 392–406, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55986/1/20693_ftp.pd

Inhibition of telomerase activity by HDV ribozyme in cancers

<p>Abstract</p> <p>Background</p> <p>Telomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity.</p> <p>Methods</p> <p>A pseudo-knotted HDV ribozyme (g.RZ57) directed against the RNA component of human telomerase (hTR) was designed and synthesized. An in vitro transcription plasmid and a eukaryotic expression plasmid of ribozyme were constructed. The eukaryotic expression plasmid was induced into heptocellular carcinoma 7402 cells, colon cancer HCT116 cells and L02 hepatocytes respectively. Then we determine the cleavage activity of ribozyme against human telomerase RNA component (hTR) both in vitro and in vivo, and detect telomerase activity continuously.</p> <p>Results</p> <p>HDV ribozyme showed a specific cleavage activity against the telomerase RNA in vitro. The maximum cleavage ratio reached about 70.4%. Transfection of HDV ribozyme into 7402 cells and colon cancer cells HCT116 led to growth arrest and the spontaneous apoptosis of cells, and the telomerase activity dropped to 10% of that before.</p> <p>Conclussion</p> <p>HDV ribozyme (g.RZ57) is an effective strategy for gene therapy.</p

Focus on function: Single molecule RNA enzymology

The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 302–316, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57325/1/20819_ftp.pd

Importance of RNA-protein interactions in bacterial ribonuclease P structure and catalysis

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) complex that catalyzes the metal-dependent maturation of the 5′ end of precursor tRNAs (pre-tRNAs) in all organisms. RNase P is comprised of a catalytic RNA (P RNA), and at least one essential protein (P protein). Although P RNA is the catalytic subunit of the enzyme and is active in the absence of P protein under high salt concentrations in vitro, the protein is still required for enzyme activity in vivo. Therefore, the function of the P protein and how it interacts with both P RNA and pre-tRNA have been the focus of much ongoing research. RNA-protein interactions in RNase P serve a number of critical roles in the RNP including stabilizing the structure, and enhancing the affinity for substrates and metal ions. This review examines the role of RNA-protein interactions in bacterial RNase P from both structural and mechanistic perspectives. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 329–338, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57327/1/20846_ftp.pd

Der wettbewerbsrechtliche Leistungsschutz | Ein Beitrag zum wettbewerbsrechtlichen Schutz vor der Ausbeutung fremder Leistungen durch das Einschieben in eine fremde Produktserie

Frank Gondert behandelt die in einer Marktwirtschaft ebenso zentrale wie grundlegende Thematik, inwieweit Nachahmung zulässig und ab wann Nachahmungsschutz zu gewähren ist. Er geht dabei der Frage nach, was Wettbewerb ist, und erörtert, inwiefern bei der Auslegung namentlich des Gesetzes gegen den unlauteren Wettbewerb in Nachahmungssachverhalten auf Erkenntnisse bezüglich des Wesens des Wettbewerbs als zugrunde liegender vorrechtlicher Regelungsmaterie zurückgegriffen werden kann und zurückzugreifen ist. Hierbei knüpft er an die Normen des UWG und des sondergesetzlichen Leistungsschutzes an und entwickelt ein eigenständiges ordnungsrechtliches Konzept, das er anwendungspraktisch vorstellt. Schließlich wird konkret ein Vorschlag zum wettbewerbsrechtlichen Schutz vor der Ausbeutung fremder Leistungen durch das Einschieben in eine fremde Produktserie entwickelt und ausdifferenziert vorgestellt

Offshore telemedicine emergency service: a 1-year experience

&lt;jats:title&gt;Abstract&lt;/jats:title&gt;&lt;jats:sec&gt; &lt;jats:title&gt;Aim&lt;/jats:title&gt; &lt;jats:p&gt;Offshore wind energy is a fast growing market. Accordingly, a correspondingly large number of employees are working at the wind farms. Owing to the harsh operating conditions, accidents and medical emergencies occur there. The care of these patients poses special challenges. The aim of the investigation was to determine whether telemedical emergency care is technically possible on the North Sea, far away from any medical care.&lt;/jats:p&gt; &lt;/jats:sec&gt;&lt;jats:sec&gt; &lt;jats:title&gt;Subject and methods&lt;/jats:title&gt; &lt;jats:p&gt;We were able to establish a raw data supported telemedical integration with a rescue service monitor for transmission of ECG, blood pressure, saturation and other vital parameters to a telemedicine centre. As a first step, a satellite connection was set up on a supply ship for the transmission, which was then made available for data transfer via WLAN.&lt;/jats:p&gt; &lt;/jats:sec&gt;&lt;jats:sec&gt; &lt;jats:title&gt;Results&lt;/jats:title&gt; &lt;jats:p&gt;In this project, we were able to show in tests as well as in actual patient care that telemedical support of rescue service personnel on site using raw data transmission is also possible offshore on a supply ship. In this project, defined areas with WLAN coverage were necessary in which the transmission worked in 100% of cases.&lt;/jats:p&gt; &lt;/jats:sec&gt;&lt;jats:sec&gt; &lt;jats:title&gt;Conclusion&lt;/jats:title&gt; &lt;jats:p&gt;The care of emergencies in the area of offshore wind farms is an increasing problem, which can be sensibly treated with telemedical support. Technical possibilities can also be created on site in the North Sea. The further expansion of a communication network, for example, with LTE or 5G, is necessary to enable telemedical care independent of supply ships.&lt;/jats:p&gt; &lt;/jats:sec&gt