Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.     

Ligand-induced stabilization of the aptamer terminal helix in the add adenine riboswitch

Riboswitches are structured mRNA elements that modulate gene expression. They undergo conformational changes triggered by highly specific interactions with sensed metabolites. Among the structural rearrangements engaged by riboswitches, the forming and melting of the aptamer terminal helix, the so-called P1 stem, is essential for genetic control. The structural mechanisms by which this conformational change is modulated upon ligand binding mostly remain to be elucidated. Here, we used pulling molecular dynamics simulations to study the thermodynamics of the P1 stem in the add adenine riboswitch. The P1 ligand-dependent stabilization was quantified in terms of free energy and compared with thermodynamic data. This comparison suggests a model for the aptamer folding in which direct P1-ligand interactions play a minor role on the conformational switch when compared with those related to the ligand-induced aptamer preorganization.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.     

Theoretical studies on the interaction of guanine riboswitch with guanine and its closest analogues

Experimental studies (M. Mandal, B. Boese, J.E. Barrick, W.C. Winkler and R.R. Breaker, Riboswitches control fundamental biochemical pathways in bacillus subtilis and other bacteria, Cell 113 (2003), pp. 577–586) demonstrated that, besides recognising guanine with high specificity, guanine riboswitch could also bind guanine analogues, but the alteration of every functionalised position on the guanine heterocycle could cause a substantial loss of binding affinity. To investigate the nature of guanine riboswitch recognising metabolites, molecular docking and molecular dynamics simulation were carried out on diverse guanine analogues. The calculation results reveal that (1) most guanine analogues could bind to guanine riboswitch at the same binding pocket, with identical orientations and dissimilar binding energies, which is related to the positions of the functional groups; (2) the two tautomers of xanthine adopt different binding modes, and the enol-tautomer shows similar binding mode and affinity of hypoxanthine, which agrees well with the experimental results and (3) the riboswitch could form stable complexes with guanine analogues by hydrogen bonding contacts with U51 and C74. Particularly, U51 plays an important role in stabilising the complexes.     

Metabolism of dimethylsulfoniopropionate and glycine betaine by a marine bacterium

Abstract The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium . MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.     

Atomic-level insights into metabolite recognition and specificity of the SAM-II riboswitch

Although S-adenosylhomocysteine (SAH), a metabolic by-product of S-adenosylmethionine (SAM), differs from SAM only by a single methyl group and an overall positive charge, SAH binds the SAM-II riboswitch with more than 1000-fold less affinity than SAM. Using atomistic molecular dynamics simulations, we investigated the molecular basis of such high selectivity in ligand recognition by SAM-II riboswitch. The biosynthesis of SAM exclusively generates the (S,S) stereoisomer, and (S,S)-SAM can spontaneously convert to the (R,S) form. We, therefore, also examined the effects of (R,S)-SAM binding to SAM-II and its potential biological function. We find that the unfavorable loss in entropy in SAM-II binding is greater for (S,S)- and (R,S)-SAM than SAH, which is compensated by stabilizing electrostatic interactions with the riboswitch. The positively charged sulfonium moiety on SAM acts as the crucial anchor point responsible for the formation of key ionic interactions as it fits favorably in the negatively charged binding pocket. In contrast, SAH, with its lone pair of electrons on the sulfur, experiences repulsion in the binding pocket of SAM-II and is enthalpically destabilized. In the presence of SAH, similar to the unbound riboswitch, the pseudoknot structure of SAM-II is not completely formed, thus exposing the Shine-Dalgarno sequence. Unlike SAM, this may further facilitate ribosomal assembly and translation initiation. Our analysis of the conformational ensemble sampled by SAM-II in the absence of ligands and when bound to SAM or SAH reveals that ligand binding follows a combination of conformational selection and induced-fit mechanisms.     

Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions

Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.     

Telomere stability and telomerase in mesenchymal stem cells

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.     

The Structure of Xis Reveals the Basis for Filament Formation and Insight into DNA Bending within a Mycobacteriophage Intasome

The recombination directionality factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein–DNA architectures. Although the attachment site DNA of mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head to tail. The structure implies a DNA bend of ~ 120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that, in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.     

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system

We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.     

Successful vitrification of bovine blastocysts,derived by in vitro maturation and fertilization

Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74–77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7–0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts. © 1993 Wiley-Liss, Inc.     

Expression of a soluble glycine binding domain of the NMDA receptor in Escherichia coli

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor. The glycine binding site of this subtype of ionotropic glutamate receptors is formed by the S1 and S2 regions of the NR1 subunit. Here, different S1S2 fusion proteins were expressed and purified from Escherichia coli cultures, and refolding protocols were established allowing the production of 30 mg of soluble S1S2 fusion protein from 1 liter bacterial culture. After affinity purification and renaturation, two of the fusion proteins (S1S2 and S1S2-V1) bound the competitive glycine site antagonist [3H]MDL105,519 with K(d) values of 9.35 and 3.9 nM, respectively. In contrast, with three other constructs (S1S2M, S1S2-V2, and -V3) saturable ligand binding could not be obtained. These results redefine the S1S2 domains required for high-affinity glycine binding. Furthermore, our high-affinity binding proteins may be used for the large-scale production of the glycine binding core region for future structural studies.     

Immunocytochemical localization of proteins P1, P2, P3 of glycine decarboxylase and of the selenoprotein PA of glycine reductase,all involved in anaerobic glycine metabolism of Eubacterium acidaminophilum

Antibodies raised against the glycine decarboxylase proteins P1, P2, P3, and the selenoprotein P_A, a component of the glycine reductase complex, were used for immunocytochemical localization experiments. Cells of Eubacterium acidaminophilum from logarithmic growth phase were fixed in the growth media with paraformaldehyde and glutaraldehyde. Fixed cells were embedded by the low-temperature procedure using Lowicryl K4M resin, and the protein A-gold technique was applied for the localization experiments. The vicinity of the cytoplasmic membrane contained about 27% of all gold particles when proteins P1 and P2 were to be localized, 50% for protein P_A, and 61% for protein P3. Double immunocytochemical labeling experiments gave evidence for the existence of a protein P1/P2 complex located predominantly in the cytoplasm, and a P3/P_A complex located at the cytoplasmic membrane. Only in very few instances the labels for proteins P3 and P1 were seen very close together in respective doublelabeling experiments. These results indicate that glycine decarboxylase does not occur in this organism as a complex consisting of all four proteins, but that protein P3, the atypical lipoamide dehydrogenase, takes part in both the glycine decarboxylase as well as in the glycine reductase reaction.     

Long-term molecular and cellular stability of human neural stem cell lines

Human Neural Stem Cells (hNSCs) are excellent candidates for in vitro and in vivo molecular, cellular, and developmental research, and also for ex-vivo gene transfer and cell therapy in the nervous system. However, hNSCs are mortal somatic cells, and thus invariably enter an irreversible growth arrest after a finite number of cell divisions in culture. It has been proposed that this is due to telomere shortening. Here, we show that long-term cultured (up to 4 years) v-myc perpetuated hNSC lines do preserve short but stable and homogeneous telomeres (TRF and Q-FISH determinations). hNSC lines (but not strains) express high levels of telomerase activity, which is activated by v-myc, as demonstrated here. Telomerase activity is not constitutive, becoming non-detectable after differentiation (in parallel to v-myc down-regulation). hNSC lines also maintain a stable cell cycle length, mitotic potential, differentiation and neuron generation capacity, and do not express senescence-associated beta-galactosidase over years, as studied here. These data, collectively, help to explain the immortal nature of v-myc-perpetuated hNSC lines, and to establish them as excellent research tools for basic and applied neurobiological and translational studies.