Jacques Vergne

The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure

The discovery of ribozymes strengthened the RNA world hypothesis, which assumes that these precursors of modern life both stored information and acted as catalysts. For the first time among extensive studies on ribozymes, we have investigated the influence of hydrostatic pressure on the hairpin ribozyme catalytic activity. High pressures are of interest when studying life under extreme conditions and may help to understand the behavior of macromolecules at the origins of life. Kinetic studies of the hairpin ribozyme self-cleavage were performed under high hydrostatic pressure. The activation volume of the reaction (34 ± 5 ml/mol) calculated from these experiments is of the same order of magnitude as those of common protein enzymes, and reflects an important compaction of the RNA molecule during catalysis, associated to a water release. Kinetic studies were also carried out under osmotic pressure and confirmed this interpretation and the involvement of water movements (78 ± 4 water molecules per RNA molecule). Taken together, these results are consistent with structural studies indicating that loops A and B of the ribozyme come into close contact during the formation of the transition state. While validating baro-biochemistry as an efficient tool for investigating dynamics at work during RNA catalysis, these results provide a complementary view of ribozyme catalytic mechanisms.

The origin of kinetic cooperativity in prehiotic catalysts

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C12) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate.Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala.A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity.It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze.

High‐pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self‐cleaving molecule

The activity of the full‐length hammerhead ribozyme requires a tertiary interaction between its distal loops leading to the closure of the molecule and its stabilization in the active conformation. In this study, the conformational changes accompanying the cis‐cleavage reaction of Chrysanthemum chlorotic mottle viroid hammerhead ribozyme were investigated by high‐pressure experiments on the complete cleavage reaction. Two activation volumes (ΔV≠) were measured, pointing to the presence of two different populations of molecules corresponding to fast‐cleaving and slow‐cleaving ribozymes in the reaction mixture. The fast population, with a small ΔV≠ of 2.6 mL·mol−1, most likely represents molecules in the near‐active conformation, whereas the slow population, with a larger ΔV≠ of 11.6 mL·mol−1, represents molecules that need a larger conformational change to induce activity. In addition, pH‐dependence experiments suggest that the group whose deprotonation is required for activity intervenes in the formation of the transition state or in the chemistry of the reaction, but not in the conformational change that precedes it.

NMR Spectroscopic Characterization of the Adenine-Dependent Hairpin Ribozyme

Time‐resolved NMR spectroscopy was applied to study ribozyme‐mediated RNA catalysis in a mutant of the hairpin ribozyme, the adenine‐dependent hairpin ribozyme (ADHR; M. Meli, et al. J. Biol. Chem. 2003, 278, 9835–9842) with atomic resolution. The mutant ADHR was designed to investigate the role of cofactors in RNA catalytic mechanisms in order to understand cellular processes that could have been present in the archaic “RNA world” and of their evolution towards functional RNAs in modern cellular processes, as for example, found in the glmS ribozyme. Conformational changes due to RNA cleavage were analyzed following spectral changes of the NMR imino proton resonances that could be assigned both for the pre‐ and postcleaved conformation for this 80‐nucleotide long RNA. 31P NMR spectroscopic studies allowed us to confirm the formation of a cyclic phosphodiester as a result of the cleavage process. For ADHR, both metal ions and the cofactor adenine are essential for self‐cleaving activity. The interaction of the ribozyme with the cofactor adenine is found to be transient and too weak to significantly change the RNA structure or to modulate the spectroscopic characteristics of the cofactor. ADHR therefore represents a ribozyme in which high activation barriers have to be overcome to populate cleavage‐competent states that exhibit short life times. We show that conformational dynamics, but not the chemistry, constitute the rate‐limiting step in catalysis of the adenine‐dependent hairpin ribozyme.

The AMINO experiment: a laboratory for astrochemistry and astrobiology on the EXPOSE-R facility of the International Space Station

The study of the evolution of organic matter subjected to space conditions, and more specifically to Solar photons in the vacuum ultraviolet range (120–200 nm) has been undertaken in low-Earth orbit since the 1990s, and implemented on various space platforms. This paper describes a photochemistry experiment called AMINO, conducted during 22 months between 2009 and 2011 on the EXPOSE-R ESA facility, outside the International Space Station. Samples with relevance to astrobiology (connected to comets, carbonaceous meteorites and micrometeorites, the atmosphere of Titan and RNA world hypothesis) have been selected and exposed to space environment. They have been analysed after return to the Earth. This paper is not discussing the results of the experiment, but rather gives a general overview of the project, the details of the hardware used, its configuration and recent developments to enable long-duration exposure of gaseous samples in tight closed cells enabling for the first time to derive quantitative results from gaseous phase samples exposed in space.

New stereoselective reaction of methylglyoxal with 2-aminopyridine and adenine derivatives: Formation of imino acid-nucleic base derivatives in water under mild conditions

A remarkable stereoselective reaction of methylglyoxal with 2-aminopyridine, the nucleic base adenine and adenine nucleosides leads in good yield to heterocycles of a new family in water under mild conditions and should be of interest in the understanding of the biological effects of methylglyoxal which is toxic, mutagenic and involved in diabetic complications.

Raman characterization of Avocado Sunblotch viroid and its response to external perturbations and self-cleavage.

BackgroundViroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest.ResultsWe report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids.ConclusionsOur results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes.

In vitro selection of adenine-dependent ribozyme against Tpl2/Cot oncogene.

Hairpin ribozymes possess the properties of RNA sequence‐specific recognition and site‐specific cleavage. These properties make them a powerful extension of the antisense approach for the inhibition of gene expression. From a randomized RNA pool of hairpin ribozymes, using the systematic evolution of ligands by exponential enrichment, we have obtained an adenine‐dependent hairpin ribozyme, Tpl2/Cot (tumour progression locus 2) ribozyme, which cleaves the Tpl2/Cot kinase mRNA sequence at nucleotides A225↓G226 relative to the start codon of translation. This serine/threonine kinase activates the mitogen‐activated protein kinase pathway implicated in cell proliferation in cancer. The selected ‘Tpl2/Cot‐YL ribozyme’ efficiently cleaves its target sequence in cis and in trans; furthermore, the ribozyme efficiently cleaves a longer target sequence of 54 nucleotides in trans, as well as the full‐length mRNA.

The AMINO experiment: RNA stability under solar radiation studied on the EXPOSE-R facility of the International Space Station

Careful examination of the present metabolism and in vitro selection of various catalytic RNAs strongly support the RNA world hypothesis as a crucial step of the origins and early life evolution. Small functional RNAs were exposed from 10 March 2009 to 21 January 2011 to space conditions on board the International Space Station in the EXPOSE-R mission. The aim of this study was to investigate the preservation or modification properties such as integrity of RNAs after space exposition. The exposition to the solar radiation has a strong degradation effect on the size distribution of RNA. Moreover, the comparison between the in-flight samples, exposed to the Sun and not exposed, indicates that the solar radiation degrades RNA bases. Received 15 April 2014, accepted 12 June 2014, first published online 18 July 2014

Hairpin Ribozyme Catalysis: A Surface-Enhanced Raman Spectroscopy Study

The existence of an “RNA world” as an early step in the history of life increases the interest for the characterization of these biomolecules. The hairpin ribozyme studied here is a self‐cleaving/ligating motif found in the minus strand of the satellite RNA associated with Tobacco ringspot virus. Surface‐enhanced Raman spectroscopy (SERS) is a powerful tool to study trace amounts of RNA. In controlled conditions, a SERS signal is proportional to the amount of free residues adsorbed on the metal surface. On RNA cleavage, residues are unpaired and free to interact with metal. SERS procedures are used to monitor and quantify the catalysis of ribozyme cleavage at biological concentrations in real time; thus, they propose an interesting alternative to electrophoretic methods.