Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

Abstract

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg²⁺ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements.

Increased concentration of Mg²⁺ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na⁺ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg²⁺.     

Conformation of free and ribosome bound rRNAs from E.coli

The effect of protein moiety on the conformation of 16S and 23S RNA of the $\text{E.}\text{coli}$ ribosome has been studied by circular dichroic spectroscopy. Both rRNAs possess a comparable net content of ordered secondary structure which remains unchanged after association with ribosomal proteins into “core” particles or into complete 30S and 50S subunits, respectively. However, differences found in the stability and the cooperativity of melting of free and protein-associated rRNAs imply protein-caused variations in the distribution of the intramolecular hairpin stems and loops and/or changes in long range tertiary interactions which appear to be different for both rRNAs. While 23S RNA is maximally stabilized on the large subunit by the full set of proteins, 16S RNA on the complete small subunit shows lower stability but higher cooperativity in melting.     

Free energy of imperfect nucleic acid helices. II. Small hairpin loops

Physical studies of enzymically synthesized oligonucleotides of defined sequence are used to evaluate quantitatively the stability of small RNA hairpin loops and helices. The series (Ap)₄G(pC) _N(pU)₄, N = 4, 5 or 6, exists as monomolecular hairpin helices when N ≥ 5, and as imperfect dimer helices when N ≤ 4. In this size range, hairpin loops become more favorable (less destabilizing thermodynamically) as they increase in size from 3 to 4 to 5 unbonded nucleotides. Very small hairpin loops are particularly destabilizing; molecules whose base sequence would imply a hairpin loop of three nucleotides will generally exist with a loop of five, including a broken terminal base pair.Thermodynamic parameters for base pair and loop formation are calculated by a method which makes unnecessary the use of measured enthalpies of polynucleotide melting. Literature data on oligonucleotide double helices yield estimates of the free energy contribution from each of the six types of stacking interactions between three possible neighboring base pairs. The advantage of this approach is that the properties of oligonucleotides are used in predicting the stability of small RNA helices, avoiding the long extrapolation from the properties of high polymers.We provide Tables of temperature-dependent free energies that allow one to predict the stability and thermal transition temperature of many simple RNA secondary structures (applicable to ~1 m-Na⁺ concentration). As an example, we apply the rules to an isolated fragment of tRNA^Ser (yeast) (Coutts, 1971), whose properties were not used in calculating the free-energy parameters. The experimental melting temperature of 88 °C is predicted with an error margin of 5 deg. C.     

Thermodynamics and kinetics of the hairpin ribozyme from atomistic folding/unfolding simulations

We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here, we introduce a simple model of tertiary-structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary-structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition-state ensemble (TSE) of the RNA during folding to its active “docked” conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters, we introduce a novel method to benchmark Monte Carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential that discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures.     

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.     

Studies of DNA dumbbells. VI. Analysis of optical melting curves of dumbbells with a sixteen-base pair duplex stem and end-loops of variable size and sequence

Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of A_t or C_t sequences (˛ = 2, 3, 4, 6, 8, 10, 14), T_t sequences (˛ = 2, 3, 4, 6, 8, 10), and G_t sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM Na⁺. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔG_loop(n > t) and single-strand circles, ΔG_cir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2_t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na⁺]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops deviate from two-state behavior.Dependence of thermodynamic parameters on[Na⁺] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by T loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.     

Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution nmr

The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNA^fMet has been examined in 0.17M NaCl, with and without Mg²⁺, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNA^fMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A₁₄–S⁴U₈ base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg²⁺ the tertiary structure base pair is present only to ～45°C, but in the presence of Mg²⁺ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A₁₄–S⁴U₈ base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg²⁺.     

Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting.

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.     

Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation

Differential scanning calorimetry (DSC), temperature-dependent uv-absorption spectroscopy, and temperature-dependent CD were used to monitor and characterize the salt-dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex-to-single-strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na⁺), a single, monophasic transition curve was observed. At lower salt (?0.01M Na⁺), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.     

Human telomerase RNA pseudoknot and hairpin thermal stability with glycine betaine and urea: preferential interactions with RNA secondary and tertiary structures

Thermal denaturation of the human telomerase RNA (hTR) DeltaU177 pseudoknot and hTR p2b hairpin was investigated by dual UV-wavelength absorbance spectroscopy in aqueous glycine betaine and urea solutions. The hTR DeltaU177 pseudoknot contains two helix-loop interactions that comprise the tertiary structure, as well as a GC-rich 6 bp stem (stem 1) and an AU-rich 9 bp stem (stem 2). The p2b hairpin also contains GC-rich stem 1 and a unique uridine-rich helix with a pentaloop. Glycine betaine stabilizes the pseudoknot tertiary structure in 135 mm NaCl and facilitates only a minor destabilization of tertiary structure in 40 mm NaCl. As with double-helical DNA, glycine betaine interacts more strongly with the surface area exposed upon unfolding of GC-rich stem 1 than either AU-rich stem 2 or the hairpin uridine-rich helix. Urea was shown to destabilize all RNA pseudoknot and hairpin secondary and tertiary structures but exhibits a stronger preferential interaction with AU-rich stem 2. Correlating these interactions with water-accessible surface area calculations indicates that the extent of interaction of glycine betaine with the surface area exposed upon RNA unfolding decreases as the nonpolar character of the unfolded RNA surface increases. As expected, the extent of interaction of urea with the surface area exposed for unfolding RNA increases as the fraction of amide functional groups increases. However, interaction of urea with amide functional groups alone cannot explain the stronger preferential interaction of urea with AU-rich stem 2. Interaction of urea with adenine relative to guanine and cytosine bases or sequence-dependent hydration is proposed for the stronger preferential interaction of urea with AU-rich duplexes.     

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.     

Thermal-unfolding Reaction of Triosephosphate Isomerase from <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate. Edgar Mixcoha-Hernández and Liliana M. Moreno-Vargas contributed equally to this work     

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.     

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.     

Cooperative Thermal Denaturation of the Assembly Origin Region of TMV RNA

Abstract

The assembly origin (AO) region of the tobacco mosaic virus RNA melts in an unusually narrow(2.5°C) temperature range. In an 0.01 M phosphate buffer the melting temperature of AO was found to be 41.5°C. This value corresponds to the regions with the most stable secondary/tertiary structure of the whole TMV RNA molecule. It is assumed that the AO region has a specific tertiary structure, which is maintained by the long-range interactions as well as by interactions of the pseudoknot type.     

Small changes in free energy assignments for unpaired bases do not affect predicted secondary structures in single stranded RNA.

We present extensive calculations of the secondary structure of mRNA which point to its insensitivity to small changes in the free energy assignments of single stranded regions. Truncating the free energies of hairpin loops, bulges, internal loops and multibranched junctions to two significant digits yields structures nearly identical to those generated using three digit values. The results show that one can safely use truncated values in RNA folding calculations. The implementation of these results enabled us to carry out secondary structure calculations on 2600 nucleotides in a single computer run.     

Structures of apomyoglobin's various acid-destabilized forms

The structures and the cold and hot melting thermodynamics of the acid- and salt-destabilized states of horse heart apomyoglobin (apoMb), including the E (extended) and various I forms, are studied using probes of tertiary structure (tryptophan fluorescence and FTIR spectroscopy) and secondary structure (far-UV CD and FTIR spectroscopy). These forms likely resemble early structures in the folding of the largely helical protein. Both the I and E forms retain the AGH core whereby the two ends of the protein are tied together with sufficient numbers of tertiary contacts, involving a number of hydrophobic residues, to show cooperative melting. The melting thermodynamics of E and I are distinctly different. E contains no other tertiary structure and probably little other secondary structure apart from the core. The more destabilized E form appears to contain "random" buried runs of polypeptide backbone which convert to alpha-helix in the I form(s). Most interestingly, E consists not of a single structure but is composed of a heterogeneous mixture of conformations, all showing corelike cooperative melting characteristics, and consisting presumably of varying contacts between the A portion of apomyoglobin and the G-H hairpin. These results bear on the energy landscape and structural features of the early part of apomyoglobin's folding pathway.     

Topological constraints are major determinants of tRNA tertiary structure and dynamics and provide basis for tertiary folding cooperativity

Recent studies have shown that basic steric and connectivity constraints encoded at the secondary structure level are key determinants of 3D structure and dynamics in simple two-way RNA junctions. However, the role of these topological constraints in higher order RNA junctions remains poorly understood. Here, we use a specialized coarse-grained molecular dynamics model to directly probe the thermodynamic contributions of topological constraints in defining the 3D architecture and dynamics of transfer RNA (tRNA). Topological constraints alone restrict tRNA''s allowed conformational space by over an order of magnitude and strongly discriminate against formation of non-native tertiary contacts, providing a sequence independent source of folding specificity. Topological constraints also give rise to long-range correlations between the relative orientation of tRNA''s helices, which in turn provides a mechanism for encoding thermodynamic cooperativity between distinct tertiary interactions. These aspects of topological constraints make it such that only several tertiary interactions are needed to confine tRNA to its native global structure and specify functionally important 3D dynamics. We further show that topological constraints are conserved across tRNA''s different naturally occurring secondary structures. Taken together, our results emphasize the central role of secondary-structure-encoded topological constraints in defining RNA 3D structure, dynamics and folding.     

Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (T_m) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol⁻¹) is considerably lower than those in the WT photoprotein (102 kcal mol⁻¹) and E179S mutant (106 kcal mol⁻¹). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.