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.     

Study of the structural organization of RNA from phage MS2 using fluorescent dyes

The spatial organization of phage MS2 RNA by binding to ethidium bromide (EtBr) and acridine orange (AO) to RNA was studied. The analyses of dye interaction by spectrophotometric and fluorometric methods have demonstrated that only about a half of 65-70% nucleotides of double-stranded segments can interact with AO and EtBr. On the other hand all the single-stranded segments appear to be accessible to AO binding. These interactions did not practically change when ionic strength (0.01-0.3), Mg2+ and Zn2+ concentrations (10(-3) M) or pH (4.7-7.4) varied. The data permit to suppose that phage MS2 RNA has a very stable tertiary structure which makes part of double-stranded segments unaccessible to inter calating dyes. Taking these and other facts into consideration we suppose that double-stranded segments play an important role in stabilization of the RNA tertiary structure. One of the most possible structure is a compact "rod-like" intramolecular aggregate of double-stranded hairpin-like segments of RNA with parallel orientation.     

Determination of base pairing in yeast 5S and 5.8S RNA infrared spectroscopy.

Infrared Spectroscopy was used to determine the numbers of base pairs for yeast 5S RNA and 5.8S RNA. The spectra were recorded at 20 degrees C and 50 degrees C, where tertiary interactions are assumed to be of less importance. It may be concluded that the structure of both RNAs is highly ordered and that there are large contributions of tertiary interactions. The results are compared with data derived from structural models that were proposed in the literature as well as with data previously published for prokaryotic 5S RNAs.     

A simple molecular model for thermophilic adaptation of functional nucleic acids

RNA molecules have numerous functions including catalysis and small molecule recognition, which typically arise from a tertiary structure. There is increasing interest in mechanisms for the thermostability of functional RNA molecules. Sosnick, Pan, and co-workers introduced the notion of "functional stability" as the free energy of the tertiary (functional) state relative to the next most stable (nonfunctional) state. We investigated the extent to which secondary structure stability influences the functional stability of nucleic acids. Intramolecularly folding DNA triplexes containing alternating T*AT and C+*GC base triples were used as a three-state model for the folding of nucleic acids with functional tertiary structures. A four-base-pair tunable region was included adjacent to the triplex-forming portion of the helix to allow secondary structure strength to be modulated. The degree of folding cooperativity was controlled by pH, with high cooperativity maintained by lower pH (5.5), and no cooperativity by higher pH (7.0). We find a linear relationship between functional free energy and the free energy of the secondary structure element adjacent to tertiary interactions, but only when folding is cooperative. We translate the definition of functional stability into equations and perform simulations of the thermodynamic data, which lend support to this model. The ability to increase the melting temperature of tertiary structure by strengthening base-pairing interactions separate from tertiary interactions provides a simple means for evolving thermostability in functional RNAs.     

Multiple folding pathways for the P4-P6 RNA domain

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.     

The three-dimensional architecture of the class I ligase ribozyme

The class I ligase ribozyme catalyzes a Mg(++)-dependent RNA-ligation reaction that is chemically analogous to a single step of RNA polymerization. Indeed, this ribozyme constitutes the catalytic domain of an accurate and general RNA polymerase ribozyme. The ligation reaction is also very rapid in both single- and multiple-turnover contexts and thus is informative for the study of RNA catalysis as well as RNA self-replication. Here we report the initial characterization of the three-dimensional architecture of the ligase. When the ligase folds, several segments become protected from hydroxyl-radical cleavage, indicating that the RNA adopts a compact tertiary structure. Ribozyme folding was largely, though not completely, Mg(++) dependent, with a K(1/2[Mg]) < 1 mM, and was observed over a broad temperature range (20 degrees C -50 degrees C). The hydroxyl-radical mapping, together with comparative sequence analyses and analogy to a region within 23S ribosomal RNA, were used to generate a three-dimensional model of the ribozyme. The predictive value of the model was tested and supported by a photo-cross-linking experiment.     

Higher order structure of chloroplastic 5S ribosomal RNA from spinach

The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.     

Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.     

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.     

Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mapping of psoralen cross-linked nucleotides in RNA

A method is described for using the cross-linking reagent 4'-(hydroxy-methyl)-4,5',8-trimethylpsoralen (HMT) to map base paired regions and higher-order structure within RNA molecules. Applying this method to yeast tRNAPhe, we have specifically identified cross-links within the acceptor stem between U6 X U68, in the D-stem between C11 X C25, and in the T psi-stem between U50 X C63 and U52 X C63. We have also identified a unique cross-link between U8 X C48 which are trans pyrimidines in the core region due to tertiary interactions between U8:A14 and C48:G15. The precise point of cross-linking was deduced in every case by using purine-specific U2 ribonuclease along with cytidine-specific CL3 ribonuclease which will anomalously cleave after photoreversed pyrimidines. The ability to map the precise point of cross-linking should prove invaluable in identifying nucleotides in close proximity within the tertiary structure of other RNA molecules.     

RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex

Tertiary interactions are critical for proper RNA folding and ribozyme catalysis. RNA tertiary structure is often condensed through long-range helical packing interactions mediated by loop-receptor motifs. RNA structures displaying helical packing by loop-receptor interactions have been solved by X-ray crystallography, but not by NMR. Here, we report the NMR structure of a 30 kDa GAAA tetraloop-receptor RNA complex. In order to stabilize the complex, we used a modular design in which the RNA was engineered to form a homodimer, with each subunit containing a GAAA tetraloop phased one helical turn apart from its cognate 11-nucleotide receptor domain. The structure determination utilized specific isotopic labeling patterns (2H, 13C and 15N) and refinement against residual dipolar couplings. We observe a unique and highly unusual chemical shift pattern for an adenosine platform interaction that reveals a spectroscopic fingerprint for this motif. The structure of the GAAA tetraloop-receptor interaction is well defined solely from experimental NMR data, shows minor deviations from previously solved crystal structures, and verifies the previously inferred hydrogen bonding patterns within this motif. This work demonstrates the feasibility of using engineered homodimers as modular systems for the determination of RNA tertiary interactions by NMR.     

Apo-parvalbumin as an intrinsically disordered protein

Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (alpha- and beta-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in alpha-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge-charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.     

Temperature dependence of benzyl alcohol- and 8-anilinonaphthalene-1-sulfonate-induced aggregation of recombinant human interleukin-1 receptor antagonist

The critical role played by temperature in ligand-induced protein aggregation was investigated. Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and the ligands benzyl alcohol and 8-anilinonaphthalene-1-sulfonate (ANS) were used. We investigated aggregation kinetics and the conformation and cysteine reactivity of rhIL-1ra in buffer alone or in the presence of 0.9% (w/v) benzyl alcohol or 4.2 or 21 mM ANS at 25 and 37 degrees C. In buffer, protein aggregation was not detected at 25 degrees C but occurred at 37 degrees C. At 25 degrees C, neither benzyl alcohol nor 4.2 mM ANS enhanced aggregation. However, at 37 degrees C, both compounds greatly accelerated protein aggregation. With 21 mM ANS, rhIL-1ra aggregation was accelerated at both temperatures, but the effect was more pronounced at 37 degrees C than at 25 degrees C. Increasing the temperature from 25 to 37 degrees C caused a minor perturbation in the tertiary structure of rhIL-1ra in buffer but no detectable alteration in secondary structure. Benzyl alcohol enhanced the tertiary structural perturbation at 37 degrees C, but the secondary structure was not affected by the ligand. The reactivity of buried free cysteines of rhIL-1ra was enhanced by benzyl alcohol at 37 degrees C but not at 25 degrees C, consistent with the structural results. Isothermal titration calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra was hydrophobic and that the degree of hydrophobic interactions increased with temperature. At 25 degrees C, the interaction of ANS with rhIL-1ra was electrostatic, but at 37 degrees C, both electrostatic and hydrophobic interactions were important. Taken together, our results support the conclusion that benzyl alcohol and ANS interact hydrophobically with partially unfolded aggregation-prone protein molecules, resulting in temperature-dependent increases in their levels and acceleration of protein aggregation.     

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg²⁺ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw^loop) of the guanine-sensing riboswitch and their Mg²⁺-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw^loop or the increase in temperature for both Gsw and Gsw^loop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg²⁺. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.     

On the role of rRNA tertiary structure in recognition of ribosomal protein L11 and thiostrepton.

Ribosomal protein L11 and an antibiotic, thiostrepton, bind to the same highly conserved region of large subunit ribosomal RNA and stabilize a set of NH4(+)-dependent tertiary interactions within the domain. In vitro selection from partially randomized pools of RNA sequences has been used to ask what aspects of RNA structure are recognized by the ligands. L11-selected RNAs showed little sequence variation over the entire 70 nucleotide randomized region, while thiostrepton required a slightly smaller 58 nucleotide domain. All the selected mutations preserved or stabilized the known secondary and tertiary structure of the RNA. L11-selected RNAs from a pool mutagenized only around a junction structure yielded a very different consensus sequence, in which the RNA tertiary structure was substantially destabilized and L11 binding was no longer dependent on NH4+. We propose that L11 can bind the RNA in two different 'modes', depending on the presence or absence of the NH4(+)-dependent tertiary structure, while thiostrepton can only recognize the RNA tertiary structure. The different RNA recognition mechanisms for the two ligands may be relevant to their different effects on protein synthesis.     

Structural and conformational stability of horseradish peroxidase: effect of temperature and pH

Detailed circular dichroism and fluorescence studies at different pHs have been carried out to monitor thermal unfolding of horseradish peroxidase isoenzyme c (HRPc). The change in CD in the 222 nm region corresponds to changes in the overall secondary structure of the enzyme, while that in the 400 nm region (Soret region) corresponds to changes in the tertiary structure around the heme in the enzyme. The temperature dependence of the tertiary structure around the heme also affected the intrinsic tryptophan fluorescence emission spectrum of the enzyme. The results suggested that melting of the tertiary structure to a pre-molten globule form takes place at 45 degrees C, which is much lower than the temperature (T(m) = 74 degrees C) at which depletion of heme from the heme cavity takes place. The melting of the tertiary structure was found to be associated with a pK(a) of approximately 5, indicating that this phase possibly involves breaking of the hydrogen-bonding network of the heme pocket, keeping the heme moiety still inside it. The stability of the secondary structure of the enzyme was also found to decrease at pH below 4.5. A 'high temperature' unfolding phase was observed which was, however, independent of pH. The stability of the secondary structure was found to drastically decrease in the presence of DTT (dithiothreitol), indicating that the 'high temperature' form is possibly stabilized due to interhelical disulfide bonds. Depletion of Ca(2+) ions resulted in a marked decrease in the stability of the secondary structure of the enzyme.     

Annotation of tertiary interactions in RNA structures reveals variations and correlations

RNA tertiary motifs play an important role in RNA folding and biochemical functions. To help interpret the complex organization of RNA tertiary interactions, we comprehensively analyze a data set of 54 high-resolution RNA crystal structures for motif occurrence and correlations. Specifically, we search seven recognized categories of RNA tertiary motifs (coaxial helix, A-minor, ribose zipper, pseudoknot, kissing hairpin, tRNA D-loop/T-loop, and tetraloop-tetraloop receptor) by various computer programs. For the nonredundant RNA data set, we find 613 RNA tertiary interactions, most of which occur in the 16S and 23S rRNAs. An analysis of these motifs reveals the diversity and variety of A-minor motif interactions and the various possible loop-loop receptor interactions that expand upon the tetraloop-tetraloop receptor. Correlations between motifs, such as pseudoknot or coaxial helix with A-minor, reveal higher-order patterns. These findings may ultimately help define tertiary structure restraints for RNA tertiary structure prediction. A complete annotation of the RNA diagrams for our data set is available at http://www.biomath.nyu.edu/motifs/.