Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.     

RNA binding small molecules: studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium

The interaction of two natural protoberberine plant alkaloids berberine and palmatine with t-RNA(phe) was studied using various biophysical techniques and the data was compared with the binding of the classical DNA intercalator, ethidium. The results of optical thermal melting, differential scanning calorimetry and circular dichroism characterized the native cloverleaf structure of t-RNA under the conditions of the study. The strong binding of the alkaloids and ethidium to t-RNA was revealed from the absorption and fluorescence studies. The salt dependence of the binding constants enabled the dissection of the binding free energy to electrostatic and non-electrostatic contributions. This analysis revealed a surprisingly large favourable component of the non-electrostatic contribution to the binding of these charged alkaloids and ethidium to t-RNA. Isothermal titration calorimetric studies revealed that the binding of both the alkaloids is driven by a moderately favourable enthalpy decrease and a moderately favourable entropy increase while that of ethidium is driven by a large favourable enthalpy decrease. Taken together, the results suggest that the binding of these alkaloid molecules on the t-RNA structure appears to be mostly by partial intercalation while ethidium intercalates to the t-RNA. These results reveal the molecular aspects on the interaction of these alkaloids to t-RNA.     

Generalized binding phenomena in an allosteric macromolecule

A general macromolecular partition function is developed in terms of chemical ligand activity, temperature and pressure for systems described by an array of species which are characterized by their state of allosteric conformation and ligand stoichiometry. The effects of chemical ligand binding, enthalpy change, and volume change are treated in a parallel manner. From a broad viewpoint all of these effects can be regarded as specific cases of generalized binding phenomena. This approach provides a general method for analyzing calorimetric and ligand binding experiments. Several applications are given: (1) Thermal scanning data for tRNAphe (P.L. Privalov and V.V. Filimonov, J. Mol. Biol. 122 (1978) 447) are shown to fit a general model with six conformational states. By application of linkage theory it is shown that sodium chloride is expelled as the molecule denatures. (2) The results of calorimetric titrations on the arabinose binding protein (H. Fukada, J.M. Sturtevant and F.A. Quiocho, J. Mol. Biol. 258 (1983) 13193) are shown to fit a simple two-state allosteric model. (3) A thermal binding curve is simulated for an unusual respiratory protein, trout I hemoglobin (B.G. Barisas and S.J. Gill, Biophys. Chem. 9 (1979) 235), in order to illustrate both the similarities and differences between enthalpy and chemical ligand binding processes.     

Replacement of RNA hairpins by in vitro selected tetranucleotides.

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA.     

Thermodynamics of ligand binding and allosteric transition in hemoglobins. Reaction of Hb trout IV with CO.

The reaction of hemoglobin trout IV with carbon monoxide has been studied in three parallel sets of experiments comprising: (1) microcalorimetric measurements in different buffers at two pH values (7.1 and 8.5) and two temperatures (20 °C and 5 °C) to determine the overall value of the enthalpy change of ligand binding as well as its point values as a function of fractional saturation with CO; (2) a study of the CO binding curves at three pH values (from 5.7 to 7.4) and at 23 °C and 5 °C in order to explore the pH and temperature dependence of the model-independent binding parameters which describe the system; (3) differential titrations in 0.2 m-sodium chloride at 23 °C and 5 °C of the deoxy and CO saturated forms of the molecule in order to determine the overall CO Bohr effect as a function of temperature. Analysis of the results of these three different sets of experiments at a purely phenomenological level shows that they are in satisfactory agreement. Further analysis in terms of a modified two-state allosteric model shows how the three model parameters k_T, k_R, and L vary with pH and temperature. From this it appears that the essential feature of the functional behavior of trout IV hemoglobin, which is characteristic of hemoglobins of teleost fish in general, lies in a proton-induced shift in the allosteric equilibrium constant. The data indicate, semiquantitatively, that the apparent enthalpy change corresponding to the allosteric equilibrium constant is pH-dependent and positive.     

Differential hydration of dA.dT base pairing and dA and dT bulges in deoxyoligonucleotides

The role of water in the formation of stable duplexes of nucleic acids is being studied by determining the concurrent volume change, heats, and counterion uptake that accompany the duplexation process. The variability of the volume contraction that we have observed in the formation of a variety of homoduplexes suggests that sequence and conformation acutely affect the degree of hydration. We have used a combination of densimetric and calorimetric techniques to measure the change in volume and enthalpy resulting from the mixing of two complementary strands to form (a) fully paired duplexes with 10 or 11 base pairs and (b) bulged decameric duplexes with an extra dA or dT unmatched residue. We also monitored absorbance vs temperature profiles as a function of strand and salt concentration for all four duplexes. Relative to the decamer duplex, insertion of an extra dA.dT base pair to form an undecamer duplex results in a favorable enthalpy of -5.6 kcal/mol that is nearly compensated by an unfavorable entropy term of -5.1 kcal/mol. This enthalpy difference correlates with a differential uptake of water molecules, corresponding to an additional hydration of 16 mol of water molecules/mol of base pair. Relative to the fully paired duplexes, both bulged duplexes are 12-16 degrees C less stable and exhibit marginally larger counterion uptake on forming the duplex. The enthalpy change is slightly lower for the T-bulge duplex and less still for the A-bulge duplex. The volume change results indicate that an unmatched residue increases the amount of coulombic and/or structural hydration. The combined results strongly suggest that the destabilizing forces in bulged duplexes are partially compensated by an increase in hydration levels.     

Crystallographic studies of RNA hairpins in complexes with recombinant MS2 capsids: implications for binding requirements.

The coat protein of bacteriophage MS2 is known to bind specifically to an RNA hairpin formed within the MS2 genome. Structurally this hairpin is built up by an RNA double helix interrupted by one unpaired nucleotide and closed by a four-nucleotide loop. We have performed crystallographic studies of complexes between MS2 coat protein capsids and four RNA hairpin variants in order to evaluate the minimal requirements for tight binding to the coat protein and to obtain more information about the three-dimensional structure of these hairpins. An RNA fragment including the four loop nucleotides and a two-base-pair stem but without the unpaired nucleotide is sufficient for binding to the coat protein shell under the conditions used in this study. In contrast, an RNA fragment containing a stem with the unpaired nucleotide but missing the loop nucleotides does not bind to the protein shell.     

Crystal structure of an RNA duplex r(gugucgcac)(2) with uridine bulges.

The crystal structure of a nonamer RNA duplex with a uridine bulge in each strand, r(gugucgcac)(2), was determined at 1.4 A resolution. The structure was solved by multiple anomalous diffraction phasing method using a three-wavelength data set collected at the Advanced Protein Source and refined to a final R(work)/R(free) of 21.2 %/23.4 % with 33,271 independent reflections (Friedel pairs unmerged). The RNA duplex crystallized in the tetragonal space group P4(1)22 with two independent molecules in the asymmetric unit. The unit cell dimensions are a=b=47.18 A and c=80.04 A. The helical region of the nonamer adopts the A-form conformation. The uridine bulges assume similar conformations, with uracils flipping out and protruding into the minor groove. The presence of the bulge induces very large twist angles (approximately +50 degrees) between the base-pairs flanking the bulges while causing profound kinks in the helix axis at the bulges. This severe twist and the large kink in turn produces a very narrow major groove at the middle of the molecule. The ribose sugars of the guanosines before the bulges adopt the C2'-endo conformation while the rest, including the bulges, are in the C3'-endo conformation. The intrastrand phosphate-phosphate (P-P) distance of the phosphate groups flanking the bulges (approximately 4.4 A) are significantly shorter than the average P-P distance in the duplex (6.0 A). This short distance between the two phosphate groups brings the non-bridging oxygen atoms close to each other where a calcium ion is bound to each strand. The calcium ions in molecule 1 are well defined while the calcium ions in molecule 2 are disordered.     

Electrostatic effects in hemoglobin: electrostatic energy associated with allosteric transition and effector binding

The pH dependence of the summed electrostatic stabilization for deoxy- and liganded hemoglobin was computed for several ionic strength values. The computed contribution to the stabilization of deoxyhemoglobin by binding of 2,3-diphosphoglycerate in the beta cleft compared well with experimental binding behavior for human hemoglobin A0 and hemoglobin F. The contribution of diphosphoglycerate binding to the alkaline Bohr effect was computed correctly for both hemoglobins A0 and F. The computed effects of simultaneous binding of diphosphoglycerate and formation of Val-1 beta carbamino adducts suggested a competition between these effectors. A direct competition was formulated between these two effectors, with extension to include a simple anion such as chloride or bicarbonate binding in competition with diphosphoglycerate but not with Val-1 beta carbamino formation. This model was found to hold at pH 7.3-7.4 over a range of concentrations of the effectors involved and to predict the pH dependence of Val-1 beta carbamino formation over the pH range 7.0-8.0. The pH dependence of the computed differential stability of liganded vs. unliganded hemoglobin A compared well with observation.     

A thermodynamic study of unusually stable RNA and DNA hairpins.

About 70% of the RNA tetra-loop sequences identified in ribosomal RNAs from different organisms fall into either (UNCG) or (GNRA) families (where N = A, C, G, or U; and R = A or G). RNA hairpins with these loop sequences form unusually stable tetra-loop structures. We have studied the RNA hairpin GGAC(UUCG)GUCC and several sequence variants to determine the effect of changing the loop sequence and the loop-closing base pair on the thermodynamic stability of (UNCG) tetra-loops. The hairpin GGAG(CUUG)CUCC with the conserved loop G(CUUG)C was also unusually stable. We have determined melting temperatures (Tm), and obtained thermodynamic parameters for DNA hairpins with sequences analogous to stable RNA hairpins with (UNCG), C(GNRA)G, C(GAUA)G, and G(CUUG)C loops. DNA hairpins with (TTCG), (dUdUCG), and related sequences in the loop, unlike their RNA counterparts, did not form unusually stable hairpins. However, DNA hairpins with the consensus loop sequence C(GNRA)G were very stable compared to hairpins with C(TTTT)G or C(AAAA)G loops. The C(GATA)G and G(CTTG)C loops were also extra stable. The relative stabilities of the unusually stable DNA hairpins are similar to those observed for their RNA analogs.     

A conformational transition involved in antagonistic substrate binding to the allosteric phosphofructokinase from Escherichia coli.

The binding of fructose 6-phosphate, ATP or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate), ADP, and phosphoenolpyruvate to Escherichia coli phosphofructokinase has been studied by changes in the protein fluorescence and/or equilibrium dialysis. The results lead to the following conclusions: (1) tetrameric phosphofructokinase can bind four ATP but only two fructose-6-phosphate, and this binding occurs without cooperativity; (2) only two conformational states, T and R, with respectively a high and a low fluorescence, seem accessible to phosphofructokinase, which exists as a mixture of one-third R and two-third T states in the absence of ligand; (3) the substrate fructose 6-phosphate and the allosteric activator ADP bind preferentially to the low-fluorescence R state, while the other substrate, ATP [or its nonhydrolyzable analogue adenylyl 5'-(beta,gamma-methylenediphosphonate)], and the allosteric inhibitor phosphoenolpyruvate bind to the high-fluorescence T state; (4) the binding of a given ligand is cooperative, with a Hill coefficient of 2, only when this binding is accompanied by a complete shift from one state to the other; for instance, the binding of the ATP analogue adenylyl 5'-(beta,gamma-methylenediphosphonate) to the T state is cooperative only in the presence of fructose 6-phosphate which favors the R state. This behavior is qualitatively consistent with a concerted transition, but quite different from that described earlier for phosphofructokinase from steady-state activity measurements (Blangy et al., 1968). This discrepancy suggests that the allosteric properties of phosphofructokinase are due in part to ligand binding and in part to the kinetics of the enzymatic reaction.     

A specific base transition occurs on replicating hepatitis delta virus RNA.

Three independent lines of evidence showed that when an infectious clone of hepatitis delta virus of known sequence was used to initiate genome replication, up to 41% of the genomes were specifically mutated in the amber termination codon (UAG to UGG) for the open reading frame of the delta antigen, thereby increasing the length of the predicted protein from 195 to 214 amino acids. This change was detected only on molecules that participated in RNA-directed RNA synthesis.     

T-T base mismatches enhance drug binding at the branch site in a four-arm DNA junction.

Base mismatches--non Watson-Crick pairing between bases--can arise in duplex DNA as a consequence of mutational events or by recombination. In a duplex, the sequence of the two bases involved, and those flanking the site of mismatch, determines the local structure and extent of destabilization of the helix. Base mismatches can arise also in recombination of nonhomologous strands, and their occurrence in Holliday recombination intermediates can influence the outcome of general or specialized recombination events. We have previously reported that the branch site in a DNA junction can interact selectively with a variety of ligands. Here we describe the thermodynamics of junctions containing T-T mismatches flanking the branch and show that these structures bind methidium and other intercalators with higher affinity than junctions lacking mismatches.     

The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.