Quinoxaline antibiotics enhance peptide nucleic acid binding to double-stranded DNA.

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.

Helical periodicity of (PNA)2.(DNA) triplexes in strand displacement complexes.

To study the helical structure in a P-loop formed by an invasion of oligopyrimidine peptide nucleic acid (PNA) into DNA duplex, bent DNA fragments containing a homopurine.homopyrimidine sequence between two bent DNA loci were prepared. As the spacer DNA length between the two bent loci varied by 1 bp over one helical turn, the electrophoretic mobility, reflecting the overall extent of DNA bending, was modulated sinusoidally in non-denaturing 5% polyacrylamide gel. When the bent DNA fragments differing in the spacer DNA length were preincubated with an oligopyrimidine PNA, the gel mobilities were changed due to a P-loop formation. By analyzing the gel mobility data with variations of the P-loop size, average helical parameters at the P-loop structure were determined. (PNA)2. (DNA) triplex within a P-loop had the helical periodicities of 15. 6(0.2) bp per turn at 20 degrees C and 17.4(0.7) bp per turn at 10 degrees C. In addition, the results indicate that a helical unwinding by 57(7) degrees at 20 degrees C and 37(13) degrees at 10 degrees C is present at the two junctions between a P-loop and its adjacent DNA duplex.

RNA folding and misfolding of the hammerhead ribozyme.

The hammerhead ribozyme undergoes a well-defined two-stage folding process induced by the sequential binding of two magnesium ions. These probably correspond to the formation of domain 2 (0-500 microM magnesium ions) and domain 1 (1-20 mM magnesium ions), respectively. In this study we have used fluorescence resonance energy transfer (FRET) to analyze the ion-induced folding of a number of variants of the hammerhead ribozyme. We find that both A14G and G8U mutations are highly destabilizing, such that these species are essentially unfolded under all conditions. Thus they appear to be blocked in the first stage of the folding process, and using uranyl-induced photocleavage we show that the core is completely accessible to this probe under these conditions. Changes at G5 do not affect the first transition but appear to provide a blockage at the second stage of folding; this is true of changes in the sugar (removal of the 2'-hydroxyl group) and base (G5C mutation, previously studied by comparative gel electrophoresis). Arrest of folding at this intermediate stage leads to a pattern of uranyl-induced photocleavage that is changed from the wild-type, but suggests a structure less open than the A14G mutant. Specific photocleavage at G5 is found only in the wild-type sequence, suggesting that this ion-binding site is formed late in the folding process. In addition to folding that is blocked at selected stages, we have also observed misfolding. Thus the A13G mutation appears to result in the ion-induced formation of a novel tertiary structure.

Effects of diaminopurine and inosine substitutions on A-tract induced DNA curvature. Importance of the 3'-A-tract junction.

Gel migration and uranyl photoprobing have been used to study the effects of inosine and 2,6-diaminopurine (2,6-DAP) substitution on adenine-tract (A-tract) induced DNA curvature. Using a 10mer repeated sequence including five inosines we show by uranyl photoprobing that a narrow minor groove varying in phase with the helix repeat is not the cause of DNA curvature. Further, we have systematically studied by gel migration the effects on A-tract induced curvature of either single or full substitution with inosine and/or 2,6-DAP in a 5'-AAAAAGCCGC-3'sequence. DNA curvature is shown to increase when inosines are substituted for the guanosines in the sequence between the A-tracts. By comparing the effects of each monosubstitution it can be seen that when the G closest to the 3'-end of the A-tract is substituted the effect on DNA curvature is much stronger than when substitution is made at any other position. By contrast, curvature is abolished when 2,6-DAP residues are substituted for all adenines, and monosubstitution reveals that the effect of substituting a single adenine is strongest at the 3'-end of the A-tract. These results favor a model in which the curvature induced by an A-tract in DNA molecules is primarily located at the junction with the 3'-end of the A-tract, and this peculiar junction is created because the A-tract has a preference to form a non-B-DNA structure which builds up from the 5'-end.

Sequence/structure selective thermal and photochemical cleavage of yeast-tRNA(Phe) by UO(2)2+.

The uranyl(VI) ion, UO(2)2+, cleaves yeast tRNA(Phe) both thermally and photochemically. Photochemical cleavage takes place at all positions but exhibits maxima at G10, G18, G30, A38, C49 and A62. Furthermore, in the presence of stoichiometric concentrations of citrate, the cleavage is generally suppressed except that strong cleavage at positions G10 and C48-U50 persists, indicating the presence of a high-affinity metal-ion binding site. It is proposed that these photocleavage sites reflect the tertiary structure of the yeast tRNA(Phe) molecule in terms of D-loop/T-loop interaction and anticodon loop conformation and that uranyl-mediated photocleavage of RNA may be used as a probe of RNA tertiary structure, and in particular for identifying binding sites for divalent metal ions. Thus a high-affinity metal-ion binding site is inferred in the "central pocket" formed by the D-loop, and the acceptor stem.

The influence of the 2-amino group of guanine on DNA conformation. Uranyl and DNase I probing of inosine/diaminopurine substituted DNA.

The conformation of the DNA helix is supposed to be a critical element in site-specific recognition by ligands both large and small. Groove width is one important measure of the conformation which varies with the local nucleotide composition, perhaps because of the presence of a purine 2-amino group on G.C base pairs. We have probed DNA with G-->inosine (I) and/or A-->diaminopurine (DAP) substitutions to see whether the location of the purine 2-amino group can indeed affect the minor groove width. At acid pH, the reactivity towards uranyl nitrate is modulated in substituted DNA quite differently from natural DNA, consistent with a marked narrowing of the minor groove at sites of G-->I substitution and widening at sites of A-->DAP replacement. The latter exerts the dominant effect. The expected changes in conformation are equally evident in the patterns of susceptibility to DNase I cleavage, but not to hydroxyl radical attack. Nuclease cleavage is maximal in normal and substituted DNA at regions of inferred moderate groove width which are generally little affected by the nucleotide substitutions. Consistent with models of sequence-dependent cutting by DNase I we find that the presence of a purine 2-amino group on the base pair three places upstream of the cutting site has a profound influence on the rate of reaction.

Ionic interactions and the global conformations of the hammerhead ribozyme.

Here we investigate the global conformation of the hammerhead ribozyme. Electrophoretic studies demonstrate that the structure is folded in response to the concentration and type of ions present. Folding based on colinear alignment of arms II and III is suggested, with a variable angle subtended by the remaining helix I. In the probable active conformation, a small angle is subtended between helices I and II. Using uranyl photocleavage, an ion binding site has been detected in the long single-stranded region. The folded conformation could generate a preactivation of the scissile bond to permit in-line attack of the 2'-hydroxyl group, with a bound metal ion playing an integral role in the chemistry.

Peptide nucleic acid.DNA strand displacement loops as artificial transcription promoters.

Homopyrimidine peptide nucleic acids (PNAs) form loop structures when binding to complementary double-stranded DNA by strand displacement, and we now show that RNA polymerase recognizes these and initiates RNA transcription from PNA/double-stranded DNA strand displacement complexes at an efficiency comparable to that of the strong Escherichia coli lacUV5 promoter. Thus PNA targets can be considered as artificial promoters controlled positively by the corresponding PNA as a transcription factor. Our results have implications for the mechanism of action of RNA polymerase and suggest the use of PNA as specific gene activating reagents and drugs.

Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.

Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.

Characterization of promoter recognition complexes formed by CRP and CytR for repression and by CRP and RNA polymerase for activation of transcription on the Escherichia coli deoP2 promoter.

The structure of the cAMP-CRP-CytR repression complex and the cAMP CRP-RNA polymerase initiation complex at the deoP2 promoter of E. coli have been probed by DNase I and uranyl footprinting. In the CRP2-CytR complex all protein DNA-phosphate contacts at CRP-1 and CRP-2 are retained, and in addition two new minor groove contacts, ascribed to phosphate-CytR interactions, are observed at -60 between the CRP sites. The contacts are compatible with a model in which the promoter DNA is wrapped around a complex of two CRPs and one CytR. In the RNA polymerase-CRP complex, the CRP-1 phosphate contacts are almost identical to those seen in the repression complex and strong RNA polymerase contacts are seen in the -10 and in the +10 regions. Most noteworthy are minor groove contacts in the -60 region ascribed to RNA polymerase contacts upstream from the CRP. Furthermore, binding of CRP to the CRP-2 target does not seem to interfere with RNA polymerase binding. Thus, a model is suggested in which the DNA is wrapped around a complex of RNA polymerase and one CRP. Finally, the results show that CytR and RNA polymerase are rivals that compete for binding with CRP at deoP2 and that CytR functions as an antiactivator.

The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12.

We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) CytR selectively regulated cAMP-CRP-dependent initiations, although transcription started from the same site in deoCp2 in the absence or presence of cAMP-CRP; (ii) deletion of the uppermost cAMP-CRP target (CRP-2) resulted in loss of CytR regulation, but had only a minor effect on positive control by the cAMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets are required for efficient CytR repression of deoCp2. Models for the action of CytR are discussed in light of these findings.

Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli.

We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the -35 region, and is sufficient for activation; the second site, CRP-2, centred around -93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro. These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP-CRP complexes.

DNA conformational analysis in solution by uranyl mediated photocleavage.

Uranyl mediated photocleavage of double stranded DNA is proposed as a general probing for DNA helix conformation in terms of minor groove width/electronegative potential. Specifically, it is found that A/T-tracts known to constitute strong distamycin binding sites are preferentially photocleaved by uranyl in a way indicating strongest uranyl binding at the center of the minor groove of the AT-region. The A-tracts of kinetoplast DNA show the highest reactivity at the 3'-end of the tract--as opposed to cleavage by EDTA/Fell--in accordance with the minor groove being more narrow at this end. Finally, uranyl photocleavage of the internal control region (ICR) of the 5S-RNA gene yields a cleavage modulation pattern fully compatible with that obtained by DNase I which also--in a more complex way--senses DNA minor groove width.

Uranyl photoprobing of conformational changes in DNA induced by drug binding.

The effect on DNA conformation upon binding of the bis-intercalator, N,N'-diacridinyl spermidine is studied by uranyl mediated DNA photocleavage and gel retardation. It is shown that a characteristic A-tract correlated 10 base pair modulation of the uranyl photocleavage of bent (kinetoplast) DNA is suppressed upon binding of the diacridine. Likewise, the anomalous slow gel electrophoretic migration of bent-DNA is abolished by diacridine binding. At higher diacridine/DNA ratios a DNA conformation in which G-residues become hypersensitive to uranyl photocleavage is induced.