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1.  Raman characterization of Avocado Sunblotch viroid and its response to external perturbations and self-cleavage 
BMC Biophysics  2014;7:2.
Viroids 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.
We 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.
Our 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.
PMCID: PMC3994434  PMID: 24655924
Viroid; RNA conformation; Self-cleavage activity; D2O perturbation; Temperature unfolding; Raman spectroscopy
2.  Structural Analyses of Avocado sunblotch viroid Reveal Differences in the Folding of Plus and Minus RNA Strands 
Viruses  2014;6(2):489-506.
Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle.
PMCID: PMC3939467  PMID: 24481250
Avocado sunblotch viroid; Ribozyme; RNA structure; SHAPE; Avsunviroidae
3.  Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium 
The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.
PMCID: PMC3264254  PMID: 22123696
4.  Replication of Avocado Sunblotch Viroid in the Yeast Saccharomyces cerevisiae▿  
Journal of Virology  2011;85(7):3229-3238.
Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. Here, we investigate the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the viroid monomeric RNA is destabilized by the nuclear 3′ and the cytoplasmic 5′ RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd.
PMCID: PMC3067865  PMID: 21270165
5.  Montmorillonite protection of an UV-irradiated hairpin ribozyme: evolution of the RNA world in a mineral environment 
BMC Evolutionary Biology  2007;7(Suppl 2):S2.
The hypothesis of an RNA-based origin of life, known as the "RNA world", is strongly affected by the hostile environmental conditions probably present in the early Earth. In particular, strong UV and X-ray radiations could have been a major obstacle to the formation and evolution of the first biomolecules. In 1951, J. D. Bernal first proposed that clay minerals could have served as the sites of accumulation and protection from degradation of the first biopolymers, providing the right physical setting for the evolution of more complex systems. Numerous subsequent experimental studies have reinforced this hypothesis.
The ability of the possibly widespread prebiotic, clay mineral montmorillonite to protect the catalytic RNA molecule ADHR1 (Adenine Dependent Hairpin Ribozyme 1) from UV-induced damages was experimentally checked. In particular, the self-cleavage reaction of the ribozyme was evaluated after UV-irradiation of the molecule in the absence or presence of clay particles. Results obtained showed a three-fold retention of the self-cleavage activity of the montmorillonite-protected molecule, with respect to the same reaction performed by the ribozyme irradiated in the absence of the clay.
These results provide a suggestion with which RNA, or RNA-like molecules, could have overcame the problem of protection from UV irradiation in the RNA world era, and suggest that a clay-rich environment could have favoured not only the formation of first genetic molecules, but also their evolution towards increasingly complex molecular organization.
PMCID: PMC1963481  PMID: 17767730
6.  Bile Salts Modulate Expression of the CmeABC Multidrug Efflux Pump in Campylobacter jejuni 
Journal of Bacteriology  2005;187(21):7417-7424.
CmeABC, a multidrug efflux pump, is involved in the resistance of Campylobacter jejuni to a broad spectrum of antimicrobial agents and is essential for Campylobacter colonization in animal intestine by mediating bile resistance. Previously, we have shown that expression of this efflux pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in the cmeABC promoter (PcmeABC) region derepresses cmeABC, leading to overexpression of this efflux pump. However, it is unknown if the expression of cmeABC can be conditionally induced by the substrates it extrudes. In this study, we examined the expression of cmeABC in the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression of cmeABC, bile salts drastically elevated the expression of this efflux operon. The induction was observed with both conjugated and unconjugated bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that bile salts inhibited the binding of CmeR to PcmeABC, suggesting that bile compounds are inducing ligands of CmeR. The interaction between bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to PcmeABC. Bile did not affect the transcription of cmeR, indicating that altered expression of cmeR is not a factor in bile-induced overexpression of cmeABC. In addition to the CmeR-dependent induction, some bile salts (e.g., taurocholate) also activated the expression of cmeABC by a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of bile salts in culture media resulted in increased resistance of Campylobacter to multiple antimicrobials. These findings reveal a new mechanism that modulates the expression of cmeABC and further support the notion that bile resistance is a natural function of CmeABC.
PMCID: PMC1272998  PMID: 16237025
7.  The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure 
Nucleic Acids Research  2005;33(8):2557-2564.
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.
PMCID: PMC1088306  PMID: 15870387

Results 1-7 (7)