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.     

Interactions of molecules with nucleic acids. IV. Binding energies and conformations of acridine and phenanthridine compounds in the two principal and in several unconstrained dimer-duplex intercalation sites

The binding positions and relative minimum binding energies are calculated for complexes of 9-aminoacridine, proflavine, N-methylphenanthridinium, and ethidium in theoretically determined intercalation sites in B-DNA (sites I and II) and in unconstrained dimer-duplex sites. The selection of site I in B-DNA by these compounds agrees with the theoretical interpretation of studies of unwinding angles in closed circular DNA in all cases but ethidium, which is predicted to select site II. The most stable binding positions of the acridines and ethidium in unconstrained dimer-duplex units agree with experimental results of intercalation complexes of dinucleoside monophosphate units. Base-pair specificity for Watson-Crick pairing is examined. The energy of an intercalation complex is partitioned into ΔE₂₃, the energy required to open base pairs BP₂ and BP₃ in B-DNA to a site, and ΔE_In, the energy change when a free molecular intercalates. ΔE₂₃ depends strongly on the base-pair sequence, whereas ΔE_In for the four molecules studied does not. The three most stable sequences contain (pyrimidine)p(purine) units, and this provides a rationale for the exclusive formation of crystals of intercalation complexes with these units. In spite of this selectivity, the distribution of G?C and A?T base pairs is equal for these three units and persists as the more unstable sequences are included. Therefore, specificity arises from the interaction between the base pairs and the 2′-deoxyribose 5′-monophosphate backbone for the opening of B-DNA to an intercalation site and not from the interaction between the chromophore and the DNA.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.     

X-ray crystallographic observation of "in-line" and "adjacent" conformations in a bulged self-cleaving RNA/DNA hybrid

The RNA strand in an RNA/DNA duplex with unpaired ribonucleotides can undergo self-cleavage at bulge sites in the presence of a variety of divalent metal ions (Hüsken et al., Biochemistry, 1996, 35:16591-16600). Transesterification proceeds via an in-line mechanism, with the 2'-OH of the bulged nucleotide attacking the 3'-adjacent phosphate group. The site-specificity of the reaction is most likely a consequence of the greater local conformational freedom of the RNA backbone in the bulge region. A standard A-form backbone geometry prohibits formation of an in-line arrangement between 2'-oxygen and phosphate. However, the backbone in the region of an unpaired nucleotide appears to be conducive to an in-line approach. Therefore, the bulge-mediated phosphoryl transfer reaction represents one of the simplest RNA self-cleavage systems. Here we focus on the conformational features of the RNA that underlie site-specific cleavage. The structures of an RNA/DNA duplex with single ribo-adenosyl bulges were analyzed in two crystal forms, permitting observation of 10 individual conformations of the RNA bulge moiety. The bulge geometries cover a range of relative arrangements between the 2'-oxygen of the bulged nucleotide and the P-O5' bond (including adjacent and near in-line) and give a detailed picture of the conformational changes necessary to line up the 2'-OH nucleophile and scissile bond. Although metal ions are of crucial importance in the catalysis of analogous cleavage reactions by ribozymes, it is clear that local strain or conformational flexibility in the RNA also affect cleavage selectivity and rate (Soukup & Breaker, RNA, 1999, 5:1308-1325). The geometries of the RNA bulges frozen out in the crystals provide snapshots along the reaction pathway prior to the transition state of the phosphoryl transfer reaction.     

The structure of helix III in Xenopus oocyte 5 S rRNA: an RNA stem containing a two-nucleotide bulge

The solution structure of an oligonucleotide containing the helix III sequence from Xenopus oocyte 5 S rRNA has been determined by NMR spectroscopy. Helix III includes two unpaired adenosine residues, flanked on either side by G:C base-pairs, that are required for binding of ribosomal protein L5. The consensus conformation of helix III in the context provided by this oligonucleotide has the two adenosine residues located in the minor groove and stacked upon the 3' flanking guanosine residue, consistent with biochemical studies of free 5 S rRNA in solution. A distinct break in stacking that occurs between the first adenosine residue of the bulge and the flanking 5' guanosine residue exposes the base of the adenosine residue in the minor groove and the base of the guanosine residue in the major groove. The major groove of the helix is widened at the site of the unpaired nucleotides and the helix is substantially bent; nonetheless, the G:C base-pairs flanking the bulge are intact. The data indicate that there may be conformational heterogeneity centered in the bulge region. The corresponding adenosine residues in the Haloarcula marismortui 50 S ribosomal subunit form a dinucleotide platform, which is quite different from the motif seen in solution. Thus, the conformation of helix III probably changes when 5 S rRNA is incorporated into the ribosome.     

Selective strand scission by intercalating drugs at DNA bulges

A bulge is an extra, unpaired nucleotide on one strand of a DNA double helix. This paper describes bulge-specific strand scission by the DNA intercalating/cleaving drugs neocarzinostatin chromophore (NCS-C), bleomycin (BLM), and methidiumpropyl-EDTA (MPE). For this study we have constructed a series of 5'-32P end labeled oligonucleotide duplexes that are identical except for the location of a bulge. In each successive duplex of the series, a bulge has been shifted stepwise up (from 5' to 3') one strand of the duplex. Similarly, in each successive duplex of the series, sites of bulge-specific scission and protection were observed to shift in a stepwise manner. The results show that throughout the series of bulged duplexes NCS-C causes specific scission at a site near a bulge, BLM causes specific scission at a site near a bulge, and MPE-Fe(II) causes specific scission centered around the bulge. In some sequences, NCS-C and BLM each cause bulge-specific scission at second sites. Further, bulged DNA shows sites of protection from NCS-C and BLM scission. The results are consistent with a model of bulged DNA with (1) a high-stability intercalation site at the bulge, (2) in some sequences, a second high-stability intercalation site adjacent to the first site, and (3) two sites of relatively unstable intercalation that flank the two stable intercalation sites. On the basis of our results, we propose a new model of the BLM/DNA complex with the site of intercalation on the 3' side (not in the center) of the dinucleotide that determines BLM binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of drug-nucleic acid interactions at atomic resolution. VII. Structure of an ethidium/dinucleoside monophosphate crystalline complex, ethidium: uridylyl(3'-5') adenosine

Ethidium forms a crystalline complex with the dinucleoside monophosphate, uridylyl (3'-5') adenosine (UpA). The complex crystallizes in the monoclinic space group P2l with unit cell dimensions, a = 13.704 A, b = 31.674 A, c = 15.131 A, beta = 113.9 degrees. This light atom structure has been solved to atomic resolution and refined by full matrix least squares to a residual of 0.12, using 3,034 observed reflections. The asymmetric unit consists of two ethidium molecules, two UpA molecules and 19 solvent molecules, a total of 145 non-hydrogen atoms. The two UpA molecules are hydrogen-bonded together by Watson-Crick type base pairing. Base-pairs in this duplex are separated by 6.7 A; this reflects intercalative binding by one of the ethidium molecules. The other ethidium molecule stacks on either side of the intercalated base-paired dinucleoside monophosphate, being related by a unit cell translation along the a axis. The conformation of the sugar-phosphate backbone accompanying intercalation has been accurately determined in this analysis, and contains the mixed sugar-puckering pattern: C3' endo (3'-5') C2' endo. This same structural feature has been observed in the ethidium-iodoUpA and ethidium-iodoCpG complexes, and exists in two additional structures containing ethidium-CpG. Taken together, these studies confirm our earlier sugar-puckering assignments and demonstrate that iodine covalently bound to the C5 position on uridine or cytosine does not alter the basic sugar-phosphate geometry or the mode of ethidium intercalation in these model studies. We have proposed this stereochemistry to explain the intercalation of ethidium (as well as other simple intercalators) into both DNA and into double-helical RNA, and discuss this aspect of our work further in this paper and in the accompanying papers.     

Bulged adenosine influence on the RNA duplex conformation in solution

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.     

Ribonuclease P RNA: models of the 15/16 bulge from Escherichia coli and the P15 stem loop of Bacillus subtilis.

The Escherichia coli ribonuclease P RNA 15/16 internal bulge loop and the Bacillus subtilis P15 stem loop are important substrate binding sites for the CCA-3' terminus of pre-tRNA. Models of E. coli 15/16 bulge loop and the B. subtilis P15 stem loop have been constructed using MC-SYM, a constraint satisfaction program. The models use covariation analysis data for suggesting initial base pairings, chemical probing, and protection/modification results to determine particular pairing orientations, and mutational experimental analysis data for tRNA-RNase P RNA contacts. The structures from E. coli and B. subtilis, although different in secondary structure, have similar sequence and function. Using MC-SYM, we are able to illustrate how the 3' end of the pre-tRNA is able to interact with this segment of the catalytic RNase P RNA. In addition, we propose additional hydrogen bonding between A76 in the 3' terminus of the tRNA and the 15/16 region of E. coli and to the loop of B. subtilis.     

Cooperative binding of R17 coat protein to RNA.

The binding of the R17 coat protein to synthetic RNAs containing one or two coat protein binding sites was characterized by using nitrocellulose filter and gel-retention assays. RNAs with two available sites bound coat protein in a cooperative manner, resulting in a higher affinity and reduced sensitivity to pH, ionic strength, and temperature when compared with RNAs containing only a single site. The cooperativity can contribute up to -5 kcal/mol to the overall binding affinity with the greatest cooperativity found at low pH, high ionic strength, and high temperatures. Similar solution properties for the encapsidation of the related fr and f2 phage suggest that the cooperativity is due to favorable interactions between the two coat proteins bound to the RNA. This system therefore resembles an intermediate state of phage assembly. No cooperative binding was observed for RNAs containing a single site and a 5' or 3' extension of nonspecific sequence, indicating that R17 coat protein has a very low nonspecific binding affinity. Unexpectedly weak binding was observed for several RNAs due to the presence of alternative conformational states of the RNA.     

Specificity in the binding of aminoglycosides to HIV-RRE RNA.

Quantitative studies of the binding of neomycin B to RRE constructs are carried out to determine the relationship between non-Watson Crick base-paired elements in the RNA and aminoglycoside binding. The RRE region contains two unpaired domains containing a single base bulge and a bubble structure, respectively. Deletion of the single base bulge has no effect on neomycin binding as the site of aminoglycoside binding is localized to the bubble region. Converting the bubble region into an A-form duplex gradually abolishes neomycin B binding in 3-5-fold steps in affinity over a 75-fold range. Thus, the binding of aminoglycoside is favored at domains in RNA that are nonduplex in nature, but aminoglycoside binding is only graded-specific in that affinities are enhanced gradually as the structure further deviates from a duplex form. It is likely that high-affinity aminoglycoside binding does not occur in duplex RNA because the major groove is too narrow to allow for aminoglycoside access and that structural perturbations that allow widening of the groove facilitate access. However, these interactions are only graded-specific with respect to both aminoglycoside structure and RNA domain structure.     

Conformational analysis of single-base bulges in A-form DNA and RNA using a hierarchical approach and energetic evaluation with a continuum solvent model.

The analysis and prediction of non-canonical structural motifs in RNA is of great importance for an understanding of the function and design of RNA structures. A hierarchical method has been employed to generate a large variety of sterically possible conformations for a single-base adenine bulge structure in A -form DNA and RNA. A systematic conformational search was performed on the isolated bulge motif and neighboring nucleotides under the constraint to fit into a continuous helical structure. These substructures were recombined with double-stranded DNA or RNA. Energy minimization resulted in more than 300 distinct bulge conformations. Energetic evaluation using a solvation model based on the finite-difference Poisson-Boltzmann method identified three basic classes of low-energy structures. The three classes correspond to conformations with the bulge base stacked between flanking nucleotides (I), location of the bulge base in the minor groove (II) and conformations with a continuous stacking of the flanking helices and a looped out bulge base (III). For the looped out class, two subtypes (IIIa and IIIb) with different backbone geometries at the bulge site could be distinguished. The conformation of lowest calculated energy was a class I structure with backbone torsion angles close to those in standard A -form RNA. Conformations very close to the extra-helical looped out bulge structure determined by X-ray crystallography were also among the low-energy structures. In addition, topologies observed in other experimentally determined bulge structures have been found among low-energy conformers. The implicit solvent model was further tested by comparing an uridine and adenine bulge flanked by guanine:cytosine base-pairs, respectively. In agreement with the experimental observation, a looped out form was found as the energetically most favorable form for the uridine bulge and a stacked conformation in case of the adenine bulge. The inclusion of solvation effects especially electrostatic reaction field contributions turned out to be critically important in order to select realistic low-energy bulge structures from a large number of sterically possible conformations. The results indicate that the approach might be useful to model the three-dimensional structure of non-canonical motifs embedded in double-stranded RNA, in particular, to restrict the number of possible conformations to a manageable number of conformers with energies below a certain threshold.     

The structure of the aflatoxin B1-DNA adduct at N7 of guanine. Theoretical intercalation and covalent adduct models

Two theoretical models are proposed for the conformational structure of both intercalated and covalent adduct complexes of aflatoxin B1, designated AFB1, with N7 of guanine of DNA. The covalent adduct model requires the DNA to kink a minimum of 39 degrees about the covalent site of the C8 and N7 atoms comprising the bond of the covalent complex. The preference of AFB1 for specific G bases within a sequence of GC content followed that of experimental studies with the added feature that for binding to the third G base of a tetramer sequence from the 3'-end, the AFB1 displayed enhanced binding at the 3' site of the targeted guanosine. Binding of AFB1 to the second G base of a tetramer sequence from the 3'-end leads to preference for a 5' site of the targeted guanosine. Inhibition of AFB1's interaction with the targeted DNA in the presence of intercalated ethidium bromide is explained by these proposed models.     

Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag)

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.     

Harmonic dynamics of a DNA hexamer in the absence and presence of the intercalator ethidium

Vibrational normal mode calculations are presented for a DNA hexanucleoside pentaphosphate, d(CpGpCpGpCpG)2, and for its complex with the cationic intercalator ethidium. Two intercalation sites are modeled that differ in DNA backbone torsion angles. Normal mode frequencies for the DNA fragment itself are significantly lower than those reported earlier using different force fields, but an analysis of "effective" frequencies suggests that somewhat higher frequencies are more appropriate. Intercalation leads to significant lowering of mobility for the base pairs adjacent to the drug; in this sequence, the ethidium binding affects the guanosine atoms more strongly than the cytosine atoms. Motions of the bases and the intercalator are analyzed in terms of "twist" about the local helix axis and a "tilt" angle relative to this axis, and the results are compared to fluorescence studies of ethidium-DNA complexes.     

Structural insights into SRP RNA: an induced fit mechanism for SRP assembly

Proper assembly of large protein-RNA complexes requires sequential binding of the proteins to the RNA. The signal recognition particle (SRP) is a multiprotein-RNA complex responsible for the cotranslational targeting of proteins to biological membranes. Here we describe the crystal structure at 2.6-A resolution of the S-domain of SRP RNA from the archeon Methanococcus jannaschii. Comparison of this structure with the SRP19-bound form reveals the nature of the SRP19-induced conformational changes, which promote subsequent SRP54 attachment. These structural changes are initiated at the SRP19 binding site and transmitted through helix 6 to looped-out adenosines, which form tertiary RNA interaction with helix 8. Displacement of these adenosines enforces a conformational change of the asymmetric loop structure in helix 8. In free RNA, the three unpaired bases A195, C196, and C197 are directed toward the helical axis, whereas upon SRP19 binding the loop backbone inverts and the bases are splayed out in a conformation that resembles the SRP54-bound form. Nucleotides adjacent to the bulged nucleotides seem to be particularly important in the regulation of this loop transition. Binding of SRP19 to 7S RNA reveals an elegant mechanism of how protein-induced changes are directed through an RNA molecule and may relate to those regulating the assembly of other RNPs.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

The influence of the local sequence environment on RNA loop structures

RNA folding is assumed to be a hierarchical process. The secondary structure of an RNA molecule, signified by base-pairing and stacking interactions between the paired bases, is formed first. Subsequently, the RNA molecule adopts an energetically favorable three-dimensional conformation in the structural space determined mainly by the rotational degrees of freedom associated with the backbone of regions of unpaired nucleotides (loops). To what extent the backbone conformation of RNA loops also results from interactions within the local sequence context or rather follows global optimization constraints alone has not been addressed yet. Because the majority of base stacking interactions are exerted locally, a critical influence of local sequence on local structure appears plausible. Thus, local loop structure ought to be predictable, at least in part, from the local sequence context alone. To test this hypothesis, we used Random Forests on a nonredundant data set of unpaired nucleotides extracted from 97 X-ray structures from the Protein Data Bank (PDB) to predict discrete backbone angle conformations given by the discretized η/θ-pseudo-torsional space. Predictions on balanced sets with four to six conformational classes using local sequence information yielded average accuracies of up to 55%, thus significantly better than expected by chance (17%-25%). Bases close to the central nucleotide appear to be most tightly linked to its conformation. Our results suggest that RNA loop structure does not only depend on long-range base-pairing interactions; instead, it appears that local sequence context exerts a significant influence on the formation of the local loop structure.