Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA: II. Structural interpretation of the thermal unfolding patterns for lupin seeds and wheat germ.

Thermal unfolding of 5S rRNA from wheat germ (WG) and lupin seeds (LS) was studied in solution. Experimental curves of differential scanning calorimetry (DSC) were resolved into particular components according to the thermodynamic model of two-state transitions. The DSC temperature profiles for WG and LS differ significantly in spite of very high similarities in the sequence of both molecules. Those results are interpreted according to a model of the secondary and tertiary molecular structure of 5S rRNA. A comparison of the 'nearest neighbour' model of interaction with the experimental thermodynamic results enables a complete interpretation of the process of the melting of its structures. In light of our observations, the crucial differences between both DSC melting profiles are mainly an outcome of different thermodynamic properties of the first helical fragment 'A' made up of 9 complementary base pairs. It contains 6 differences in the nucleotide sequence of both types of molecules, which still retain 9-meric double helixes. The temperature stability of his helix in WG is much lower than of the LS one. Moreover, the results supply evidence for a strong specific tertiary interaction between the two hairpin loops 'c' and 'e' in both 5S rRNA molecules, modulated by small differences in the thermodynamic properties of both 5S rRNA.     

84 PPC measurements of unfolding volumes of DNA stem-loop motifs

One focus of our research is to further our understanding of the physico-chemical properties of non-canonical nucleic acid structures. In this work, DNA hairpins are used to mimic a common motif present in RNA, i.e. a stem-loop motif with a bulge or internal loop in their stem. Specifically, we used a combination of temperature-dependent UV spectroscopy, differential scanning (DSC), and pressure perturbation (PPC) calorimetric techniques to determine complete thermodynamic profiles for the helix–coil transitions of two sets of hairpins with 5′–3′ sequences: d(GCGCT _n GTAACT₅GTTACGCGC) and d(GCGCT _n GTAACT₅GTTACT _n GCGC). “T _n ” is a variable loop of thymines, n?=?1, 3 or 5; and “T₅” is an end-loop of five thymines. Unfolding curves show monophasic transitions with TMs independent of strand concentration, confirming their intramolecular formation. DSC thermodynamic profiles indicate that the favorable folding of each hairpin results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, while the DSC curves as a function of salt concentration yielded an uptake of cations and negative heat capacity effects. PPC melting curves yielded positive folding volumes ranging 12–31?cm³/mol, corresponding to releases of water molecules; in contrast, an uptake of water (ranging from 32 to 63?mol of H₂O/mol) is observed from osmotic stress experiments using ethylene glycol as the osmolyte. Overall, the increase in the size of the variable bulge or internal-loop yielded lower TMs and slightly more favorable enthalpies, corresponding to less favorable free energy contributions of ～0.7?kcal/mol per thymine residue. The volume measurements will be correlated with the unfolding entropies and discussed in terms of the type of water that is hydrating these stem-loop motifs structures.     

Differential effects of ribosomal proteins and Mg2+ ions on a conformational switch during 30S ribosome 5′-domain assembly

Ribosomal protein S4 nucleates assembly of the 30S ribosome 5′ and central domains, which is crucial for the survival of cells. Protein S4 changes the structure of its 16S rRNA binding site, passing through a non-native intermediate complex before forming native S4-rRNA contacts. Ensemble FRET was used to measure the thermodynamic stability of non-native and native S4 complexes in the presence of Mg²⁺ ions and other 5′-domain proteins. Equilibrium titrations of Cy3-labeled 5′-domain RNA with Cy5-labeled protein S4 showed that Mg²⁺ ions preferentially stabilize the native S4-rRNA complex. In contrast, ribosomal proteins S20 and S16 act by destabilizing the non-native S4-rRNA complex. The full cooperative switch to the native complex requires S4, S16, and S20 and is achieved to a lesser degree by S4 and S16. The resulting thermodynamic model for assembly of the 30S body illustrates how ribosomal proteins selectively bias the equilibrium between alternative rRNA conformations, increasing the cooperativity of rRNA folding beyond what can be achieved by Mg²⁺ ions alone.     

83 Application of differential scanning calorimetry to measure the differential binding of ions,water, and protons in the unfolding of DNA molecules

The overall stability of DNA molecules globally depends on base-pair stacking, base pairing, polyelectrolyte effect, and hydration contributions. In order to improve our understanding of the role of ions, water, and protons in the stability and melting behavior of DNA structures, we report an experimental approach to determine the differential binding of ions (Δn _ion), water (Δn _W), and protons (Δn _H+) in the helix-coil transition of DNA molecules. A combination of differential scanning calorimetry (DSC) and temperature-dependent UV and CD spectroscopic techniques to investigate the unfolding of a variety of DNA molecules: S.T. DNA, two dodecamers, one undecamer, nine short hairpins as a function of the GC content of their stem, and two triplexes. We determine complete thermodynamic profiles, including all the three linking numbers, for the unfolding of each molecule. The CD spectra indicated that all molecules adopted the B-conformation at low temperatures. Thermodynamic profiles obtained from the DSC curves indicate that the favorable folding of each molecule results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, and negligible heat capacity effects. UV and DSC melting curves as a function of salt, osmolyte, and proton concentrations yielded releases of ions, water, and protons (for the triplex with C⁺GC base triplets). Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of water and counterions. The thermodynamic data will be discussed in terms of the effects of DNA length, loop contributions and type of water molecules.     

Temperature‐induced partially unfolded state of hUBF HMG Box‐5: Conformational and dynamic investigations of the Box‐5 thermal intermediate ensemble

Human upstream binding factor (hUBF) HMG Box‐5 is a highly conserved protein domain, containing 84 amino acids and belonging to the family of the nonspecific DNA‐binding HMG boxes. Its native structure adopts a twisted L shape, which consists of three α‐helices and two hydrophobic cores: the major wing and the minor wing. In this article, we report a reversible three‐state thermal unfolding equilibrium of hUBF HMG Box‐5, which is investigated by differential scanning calorimetry (DSC), circular dichroism spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. DSC data show that Box‐5 unfolds reversibly in two separate stages. Spectroscopic analyses suggest that different structural elements exhibit noncooperative transitions during the unfolding process and that the major form of the Box‐5 thermal intermediate ensemble at 55°C shows partially unfolded characteristics. Compared with previous thermal stability studies of other boxes, it appears that Box‐5 possesses a more stable major wing and two well separated subdomains. NMR chemical shift index and sequential ¹H^N_i‐¹H^N_i+1 NOE analyses indicate that helices 1 and 2 are native‐like in the thermal intermediate ensemble, while helix 3 is partially unfolded. Detailed NMR relaxation dynamics are compared between the native state and the intermediate ensemble. Our results implicate a fluid helix‐turn‐helix folding model of Box‐5, where helices 1 and 2 potentially form the helix 1‐turn‐helix 2 motif in the intermediate, while helix 3 is consolidated only as two hydrophobic cores form to stabilize the native structure. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration

We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2′-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix–coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2′-deoxyuridine (dU*)], and the interaction of each hairpin with Mg²⁺. All three molecules undergo two-state transitions with melting temperatures (T_M) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T_M values of 64–66°C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4–6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36–39 kcal/mol, and favorable entropies of ~110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T_M-dependence on salt yielded slopes of 2.3–2.9°C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na⁺ per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg²⁺ with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2–4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.     

The 3′ Terminal Colicin Fragment of Escherichia coli 16S Ribosomal RNA. Conformational Details Revealed by Enzymic and Chemical Probing

Abstract

The conformation of the colicin fragment of E. coli 16S rRNA was probed with various nucleases and with the adenosine-specific reagent diethylpyrocarbonate (DEP). The results confirm the presence of a stable central hairpin in the colicin fragment and a weaker additional secondary structure involving the regions 5′ and 3′ to this hairpin. By monitoring DEP accessibility at various stages of heat-denaturation sequential unfolding of individual base pairs was followed.

In agreement with previous results it could be shown that dimethylation of the two adjacent adenosines in the hairpin loop (a feature in virtually all ribosomes) leads to a destabilization of the hairpin helix.

Accessibilities of G residues, involved in the weaker additional secondary structure is anomalous. One G residue is sensitive to the single strand specific RN ase T₁ and insensitive to DEP, while the situation is reversed for the adjoining G residue. The strong reaction of the latter G-residue with DEP is unusual and indicates a very special conformation.     

Conformational changes in transfer RNA. I. Equilibrium theory

We report the results of semi-empirical calculations describing thermodynamic properties of transfer RNA conformations. The most important new features of the procedure are: (1) the use of parameters obtained from model oligoribonucleotides to evaluate the free energy of helical regions and small hairpin loops, and (2) the use of a model which is somewhat more realistic than the freely jointed chain for evaluating internal loops and intermediate size hairpin loops. The new parameters lead to important quantitative and qualitative differences from predictions which would have been made in the past and lead to a priori predictions of tRNA melting temperatures which are within about 6°C of the experimental values. The results suggest the following conclusions: (1) The early melting transition observed in several tRNA's is partly the result of tertiary unfolding, and partly the result of the loss of some secondary structure. (2) The part of the secondary structure which melts during the early transition is different for different tRNA's. For fMet and Tyr from E. coli, the calculations predict that the dihydrouradiene arm melts out early. For yeast Phe the acceptor stem and anticodon helix melt first. (3) The results also suggest the possibility that tertiary unfolding and early secondary structural melting do not occur independently but are coupled, so that the two types of structure are probably mutually stabilizing.     

Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

NMR structure and Mg2+ binding of an RNA segment that underlies the L7/L12 stalk in the E.coli 50S ribosomal subunit

Helix 42 of Domain II of Escherichia coli 23S ribosomal RNA underlies the L7/L12 stalk in the ribosome and may be significant in positioning this feature relative to the rest of the 50S ribosomal subunit. Unlike the Haloarcula marismortui and Deinococcus radiodurans examples, the lower portion of helix 42 in E.coli contains two consecutive G•A oppositions with both adenines on the same side of the stem. Herein, the structure of an analog of positions 1037–1043 and 1112–1118 in the helix 42 region is reported. NMR spectra and structure calculations support a cis Watson–Crick/Watson–Crick (cis W.C.) G•A conformation for the tandem (G•A)₂ in the analog and a minimally perturbed helical duplex stem. Mg²⁺ titration studies imply that the cis W.C. geometry of the tandem (G•A)₂ probably allows O6 of G20 and N1 of A4 to coordinate with a Mg²⁺ ion as indicated by the largest chemical shift changes associated with the imino group of G20 and the H8 of G20 and A4. A cross-strand bridging Mg²⁺ coordination has also been found in a different sequence context in the crystal structure of H.marismortui 23S rRNA, and therefore it may be a rare but general motif in Mg²⁺ coordination.     

The in vitro synthesis of lysozyme,total proteins,and polyphenylalanine by ribosomes containing hydrolyzed ribonucleic acid

Ribosomes containing 23S rRNA with one scission per molecule were found to be inactive in the synthesis of lysozyme, total protein, and polyphenylalanine at 9.1 mm Mg²⁺. Increasing the Mg²⁺ concentration to 12.0 mm restored synthesis of lysozyme and total proteins. Ribosomes with two or more scissions in 23S rRNA were fully active in the synthesis of lysozyme, total protein, and polyphenylalanine at 9.1 mm Mg²⁺. It appears that one scission in the 23S rRNA molecule in a 70S ribosome allows the structure of the ribosome to change so as to disorient ribosomal proteins or rRNA. A second scission in 23S rRNA or an increase in Mg²⁺ concentration reverses the change which occurred with the first scission.     

Mg2+ Impacts the Twister Ribozyme through Push-Pull Stabilization of Nonsequential Phosphate Pairs

RNA molecules perform a variety of biological functions for which the correct three-dimensional structure is essential, including as ribozymes where they catalyze chemical reactions. Metal ions, especially Mg²⁺, neutralize these negatively charged nucleic acids and specifically stabilize RNA tertiary structures as well as impact the folding landscape of RNAs as they assume their tertiary structures. Specific binding sites of Mg²⁺ in folded conformations of RNA have been studied extensively; however, the full range of interactions of the ion with compact intermediates and unfolded states of RNA is challenging to investigate, and the atomic details of the mechanism by which the ion facilitates tertiary structure formation is not fully known. Here, umbrella sampling combined with oscillating chemical potential Grand Canonical Monte Carlo/molecular dynamics simulations are used to capture the energetics and atomic-level details of Mg²⁺-RNA interactions that occur along an unfolding pathway of the Twister ribozyme. The free energy profiles reveal stabilization of partially unfolded states by Mg²⁺, as observed in unfolding experiments, with this stabilization being due to increased sampling of simultaneous interactions of Mg²⁺ with two or more nonsequential phosphate groups. Notably, these results indicate a push-pull mechanism in which the Mg²⁺-RNA interactions actually lead to destabilization of specific nonsequential phosphate-phosphate interactions (i.e., pushed apart), whereas other interactions are stabilized (i.e., pulled together), a balance that stabilizes unfolded states and facilitates the folding of Twister, including the formation of hydrogen bonds associated with the tertiary structure. This study establishes a better understanding of how Mg²⁺-ion interactions contribute to RNA structural properties and stability.     

Solution conformations of B. subtilis ribosomal 5S RNA: a calorimetric study

The unfolding of B. subtilis 5S RNA is examined by direct calorimetric measurement in the presence of various concentrations of Na⁺ and Mg²⁺. The composite differential scanning calorimetry (DSC) curve is analyzed into 3–5 individual two-state melting transitions. In the absence of added Na⁺ or Mg²⁺, the 5S RNA segments melt together at T_m = 40°C. Addition of Na⁺ stabilizes the molecular structure (T_m = 56°C) and widens the melting temperature range, so that up to five component transitions are observed. Addition of Mg²⁺ alone produces a very stable structure (T_m = 75°C) with highly cooperative melting. Finally, addition of both Na⁺ and Mg²⁺ produces the highest stability (T_m = 76°C). The results are interpreted according to hypothetical secondary and tertiary base-pairing schemes. The conformational changes demonstrated here may facilitate the movement of the protein synthesis machinery during RNA translation.     

Solution structures of polcalcin Phl p 7 in three ligation states: Apo‐, hemi‐Mg2+‐bound,and fully Ca2+‐bound

Polcalcins are small EF‐hand proteins believed to assist in regulating pollen‐tube growth. Phl p 7, from timothy grass (Phleum pratense), crystallizes as a domain‐swapped dimer at low pH. This study describes the solution structures of the recombinant protein in buffered saline at pH 6.0, containing either 5.0 mM EDTA, 5.0 mM Mg²⁺, or 100 μM Ca²⁺. Phl p 7 is monomeric in all three ligation states. In the apo‐form, both EF‐hand motifs reside in the closed conformation, with roughly antiparallel N‐ and C‐terminal helical segments. In 5.0 mM Mg²⁺, the divalent ion is bound by EF‐hand 2, perturbing interhelical angles and imposing more regular helical structure. The structure of Ca²⁺‐bound Phl p 7 resembles that previously reported for Bet v 4—likewise exposing apolar surface to the solvent. Occluded in the apo‐ and Mg²⁺‐bound forms, this surface presumably provides the docking site for Phl p 7 targets. Unlike Bet v 4, EF‐hand 2 in Phl p 7 includes five potential anionic ligands, due to replacement of the consensus serine residue at –x (residue 55 in Phl p 7) with aspartate. In the Phl p 7 crystal structure, D55 functions as a helix cap for helix D. In solution, however, D55 apparently serves as a ligand to the bound Ca²⁺. When Mg²⁺ resides in site 2, the D55 carboxylate withdraws to a distance consistent with a role as an outer‐sphere ligand. ¹⁵N relaxation data, collected at 600 MHz, indicate that backbone mobility is limited in all three ligation states. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Interaction of the dimeric form of retroviral RNA with paromonycin and magnesium as revealed using 2-aminopurine fluorescence

Studying the dimeric RNA structural organization is a step toward the understanding of retroviral genomic RNA dimerization. A kissing loop dimer is rearranged into an extended dimer during maturation of the virus particle. The extended dimer formation may be inhibited by ligands interacting with the RNA kissing loop dimer. A study was made of the interaction of dimeric RNA with paromomycin and magnesium ions. RNA dimers were formed from two hairpin RNAs having complementary sequences in the loop. The structural features of RNA dimers and the influence of the ligands were inferred from the fluorescence of 2-aminopurine (2-AP) incorporated in one of the two RNA hairpin sequences. As dimeric RNA interacted with paromomycin, 2-AP fluorescence increased. The increase was explained by a flipping of the fluorescent base out of the RNA structure. The binding constants and stoichiometry were estimated for dimeric RNA binding with paromomycin. An RNA dimer was found to interact with two paromomycin molecules; the binding constant was approximately the same (about 3 × 10⁵ M⁻¹) for both types of dimers. It was observed that the antibiotic and Mg²⁺ ions compete for binding to the hairpin RNA dimer and that one paromomycin molecule is displaced by one Mg²⁺ ion.     

Molecular beacons as probes of RNA unfolding under native conditions

Hybridization of fluorescent molecular beacons provides real-time detection of RNA secondary structure with high specificity. We used molecular beacons to measure folding and unfolding rates of the Tetrahymena group I ribozyme under native conditions. A molecular beacon targeted against 15 nt in the 5′ strand of the P3 helix specifically hybridized with misfolded forms of the ribozyme, without invading the native tertiary structure. The beacon associated with the misfolded ribozyme 300 times more slowly than with an unstructured oligonucleotide containing the same target sequence, suggesting that the misfolded ribozyme core remains structured in the absence of Mg²⁺. The rate of beacon hybridization under native conditions revealed a linear relationship between the free energy of unfolding and Mg²⁺ concentration. A small fraction of the RNA population unfolded very rapidly, suggesting parallel unfolding in one step or through misfolded intermediates.     

Sequence Heterogeneity of the Ten rRNA Operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA^Ile genes between the 16S and 23S rDNAs, and the other had a tRNA^Ile genes between the 16S and 23S rDNAs and a tRNA^Asn gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.     

Melting behavior and ligand binding of DNA intramolecular secondary structures

We use a variety of biophysical techniques to determine thermodynamic profiles, including hydration, for the unfolding of DNA stem-loop motifs (hairpin, a three-way junction and a pseudoknot) and their interaction with netropsin and random cationic copolymers. The unfolding thermodynamic data show that their helix-coil transition takes place according to their melting domains or sequences of their stems. All hairpins adopted the B-like conformation and their loop(s) contribute with an immobilization of structural water. The thermodynamic data of netropsin binding to the ^5′-AAATT-^3′/TTTAA site of each hairpin show affinities of ~ 10^6- 7 M^− 1, 1:1 stoichiometries, exothermic enthalpies of − 7 to − 12 kcal mol^− 1 (− 22 kcal mol^− 1 for the secondary site of the three-way junction), and water releases. Their interaction with random cationic copolymers yielded higher affinities of ~ 10⁶ M^− 1 with the more hydrophobic hairpins. This information should improve our current picture of how sequence and loops control the stability and melting behavior of nucleic acid molecules.