A potential RNA drug target in the hepatitis C virus internal ribosomal entry site.

Subdomain IlId from the hepatitis C virus (HCV) internal ribosome entry site (IRES) has been shown to be essential for cap-independent translation. We have conducted a structural study of a 27-nt fragment, identical in sequence to IlId, to explore the structural features of this subdomain. The proposed secondary structure of IlId is comprised of two 3 bp helical regions separated by an internal loop and closed at one end by a 6-nt terminal loop. NMR and molecular modeling were used interactively to formulate a validated model of the three-dimensional structure of IlId. We found that this fragment contains several noncanonical structural motifs and non-Watson-Crick base pairs, some of which are common to other RNAs. In particular, a motif characteristic of the rRNA alpha-sarcin/ricin loop was located in the internal loop. The terminal loop, 5'-UUGGGU, was found to fold to form a trinucleotide loop closed by a trans-wobble U.G base pair. The sixth nucleotide was bulged out to allow stacking of this U.G pair on the adjacent helical region. In vivo mutational analysis in the context of the full IRES confirmed the importance of each structural motif within IIId for IRES function. These findings may provide clues as to host cellular proteins that play a role in IRES-directed translation and, in particular, the mechanism through which host ribosomes are sequestered for viral function.

Site-specific cross-linking of amino acids in the basic region of human immunodeficiency virus type 1 Tat peptide to chemically modified TAR RNA duplexes.

The Human Immunodeficiency Virus type 1 Tat protein interacts specifically with a U-rich bulge within an RNA stem-loop known as the trans-activation responsive region (TAR) that occurs in all viral transcripts. We have photochemically cross-linked to Tat peptide (37-72), a model TAR duplex substituted at U23 in the bulge by 4-thioU. We have identified the cross-linked amino acid as Arg55 in the basic region of the Tat peptide by use of a combination of proteolytic digestions and MALDI-TOF mass spectrometric analysis. The identification also required use of a synthetic Tat peptide containing a site-specific, uniformly 13C and 15N isotopically labeled arginine. We also describe a new chemical procedure for obtaining site-specific cross-links to Tat via the use of 2'-beta-alanyl U-substituted TAR and the amino-specific reagent dithiobis(succinimidyl propionate). U23-2'-functionalized TAR was shown to cross-link uniquely to Lys51 in the basic region of Tat, whereas other sites in the upper and lower stems of TAR (U35, U38, and U42) showed cross-linking only to the N-terminus of Tat peptide (37-72). U40 cross-linked to both Lys51 and the N-terminus of the peptide. The results help to establish a preliminary model of the binding of Tat peptide to the major groove of TAR RNA.

An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication.

One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 x 10(6) individual chemical entities, we were able to select a hybrid peptoid/peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat/TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat-binding site. In addition, 10-30 microM CGP64222 specifically inhibited Tat activity in a cellular Tat-dependent transactivation assay [fusion-induced gene stimulation (FIGS) assay] and blocked HIV-1 replication in primary human lymphocytes. By contrast, peptides of a comparable size and side-chain composition inhibited cell fusion in the FIGS assay and only partially inhibited HIV-1 replication in primary human lymphocytes. Thus, we have discovered a compound, CGP64222, that specifically inhibits the Tat/TAR RNA interaction, both in vitro and in vivo.

Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.

Efficient transcription from the human immunodeficiency virus (HIV) promoter depends on binding of the viral regulatory protein Tat to a cis-acting RNA regulatory element, TAR. Tat binds at a trinucleotide bulge located near the apex of the TAR stem-loop structure. An essential feature of Tat-TAR interaction is that the protein induces a conformational change in TAR that repositions the functional groups on the bases and the phosphate backbone that are critical for specific intermolecular recognition of TAR RNA. We have previously determined a high resolution structure for the bound form of TAR RNA using heteronuclear NMR. Here, we describe a high resolution structure of the free TAR RNA based on 871 experimentally determined restraints. In the free TAR RNA, bulged residues U23 and C24 are stacked within the helix, while U25 is looped out. This creates a major distortion of the phosphate backbone between C24 and G26. In contrast, in the bound TAR RNA, each of the three residues from the bulge are looped out of the helix and U23 is drawn into proximity with G26 through contacts with an arginine residue that is inserted between the two bases. Thus, TAR RNA undergoes a transition from a structure with an open and accessible major groove to a much more tightly packed structure that is folded around basic side chains emanating from the Tat protein.

The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is introduced to the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop encoded by the 5' leader sequence found on all HIV-1 mRNAs. We have used multidimensional heteronuclear NMR to determine the structure of the TAR RNA in the presence of the ADP-1 polypeptide, a 37-mer that carries the minimal RNA recognition region of the Tat protein and closely mimics Tat binding specificity. In the presence of a variety of ligands, including ADP-1, related basic peptides and the amino acid derivative argininamide, the bulge region of TAR undergoes a local conformational rearrangement and forms a more stable structure. The structure of TAR in the bound form has been determined from over 1000 NMR-derived constraints. The U23 residue at the 5' end of the bulge is positioned near G26 and A27 in the major groove, rather than stacked on A22 as in the free TAR. U23 and G26 are brought into close proximity by contacts to the guanidinium group and side-chain amide group of a common arginine residue. However, the interaction of this guanidinium group with TAR is not the only source of binding specificity. Besides NOEs to the arginine residue participating in the conformational change, ADP-1 shows additional intermolecular NOEs to TAR, suggesting that there are multiple points of contacts between TAR RNA and residues from the basic and core regions of Tat. These structural results provide important clues towards the identification of small molecular mass and/or peptidomimetic inhibitors of the essential Tat-TAR interaction.

Novel three-dimensional 1H-13C-31P triple resonance experiments for sequential backbone correlations in nucleic acids.

Backbone-driven assignment methods that utilize covalent connectivities have greatly facilitated spectral assignments of proteins. In nucleic acids, 1H-13C-31P correlations could play a similar role, and several related experiments (HCP) have recently been presented for backbone-driven sequential assignments in RNA. The three-dimensional extension of 1H-31P Het-Cor (P,H-COSY-H,C-HMQC) and Het-TOCSY (P,H-TOCSY-H,C-HMQC) experiments presented here complements HCP experiments as tools for spectral assignments and extraction of dihedral angle constraints. By relying on 1H-31P rather than 13C-31P couplings to generate cross peaks, the strongest connectivities are observed in different spectral regions, increasing the likelihood of resolving spectral overlap. In addition, semiquantitative estimates of 1H-31P and 13C-31P couplings provide dihedral angle constraints for RNA structure determination.

Novel techniques in nuclear magnetic resonance for nucleic acids.

Recent techniques for the efficient preparation of isotopically labelled RNA of desired sequence represent a dramatic step forward for NMR of nucleic acids. Three- and four-dimensional NMR experiments greatly facilitate spectral analysis and quantification of structural constraints. Backbone-driven assignment procedures have been introduced to parallel the powerful assignment methods introduced for work with proteins. Additional structural information to complement interproton distances, namely scalar coupling constants defining the backbone conformation, can be obtained using isotopically labelled oligonucleotides. The additional interproton distance and dihedral angle constraints resolved in higher-dimensional spectra will enable the determination of larger DNA and RNA structures and also increase accuracy and precision.

Solution structure of a parallel-stranded tetraplex formed by d(TG4T) in the presence of sodium ions by nuclear magnetic resonance spectroscopy.

DNA sequences containing repetitive oligo-guanine sequences are observed in telomeric DNA and in recombination hotspots. In vitro, these sequences fold into at least two classes of unusual conformations involving the formation of guanine tetrads (G-quartets). Through the use of NMR, we have established that in solution in the presence of sodium ions, the oligodeoxynucleotide dTG4T forms a parallel-stranded tetraplex, with hydrogen-bonded guanine tetrads. Here we present the detailed evidence for this configuration in 100 mM sodium ions at neutral pH. We show that the parallel-stranded tetraplex structure is stabilised relative to the single-strands when Na ions are replaced by K ions, without any evidence of significant change in those chemical shifts associated with the tetraplex form. We also present a model for the Na+ solution structure of the tetraplex, by back-calculation from NOE volumes. We obtain excellent agreement with the data for structures with characteristics similar to B-DNA for the internal tetrads, G3 and G4. The outer tetrads, particularly the 5'-terminal G2, display some unstacking. We see no evidence for formation of thymine tetrads analogous to the uridine tetrads observed in RNA, but rather we find the terminal thymine conformations are not well determined by our data. We observe an uninterrupted cylindrical channel through the centre of the complex.

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.

Distinguishing between duplex and hairpin forms of RNA by 15N-1H heteronuclear NMR.

A general method is described for distinguishing RNA hairpins from RNA duplexes by application of two-dimensional filtered nuclear Overhauser enhancement spectra on a 1:1 mixture of unlabeled and 99% 15N-labeled molecules. The method is applied to the RNA dodecamer rGGCGCUUGCGUC which can form an intramolecular hairpin under low salt conditions and a duplex in high salt. This procedure allows unambiguous identification of RNA hairpins or duplexes under the same conditions that are used in the NMR solution structure determination.

Effect of Vicia faba and bran feeding on nitrosamine carcinogenesis and formation.

The goal of this work was to study the effect of the most common Egyptian food items, Vicia faba beans (VF) and bran, on the carcinogenicity of dibutylnitrosamine (DBN) precursors (dibutylamine and nitrite). Mice receiving DBN precursors showed a delayed gain in body weight as well as decreased protein level and 5-nucleotidase activity. Acid ribonuclease, alkaline phosphatase, and DNA level and rate of synthesis were significantly increased compared with corresponding controls. Hepatomas and bladder papillomas developed in 60% and 40% of mice, respectively, after nine months of treatment. On the other hand, administration of VF or bran, in addition to DBN precursors, lessened the damage caused by DBN precursors alone, except DNA level and rate of synthesis were elevated. Alkaline phosphatase was also elevated when bran was administered with DBN precursors. However, these elevations were still less than corresponding elevations in mice receiving DBN precursors alone. The incidence of hepatoma was also reduced to only 20% for both groups. Meanwhile, incidence of bladder papillomas was only 20% in mice receiving VF in addition to DBN precursors, and bladder papillomas were completely absent in mice receiving bran in addition to DBN precursors. In vitro studies were also performed to clarify the effect of VF or bran on diphenylnitrosamine (DPhNA) and its formation from its precursors (diphenylamine and nitrite). The study revealed that VF and bran have the ability to eliminate nitrite and DPhNA from the reaction media and to reduce the rate of formation of DPhNA from its precursors. This reaction depends on the concentration and form of VF or bran and the duration of the reaction. Thus it is concluded that some naturally occurring food items, such as VF and bran, could protect humans against the hazardous effect of nitrosamines and their precursors.

Nuclear magnetic resonance study of a deoxyoligonucleotide duplex containing a three base bulge.

The three-dimensional structures of a DNA oligonucleotide containing three extra unpaired adenosine residues (dGCCAGGAAATGGAC+dGTCCGACCTGGC) and that of the perfect duplex analogue (dGCCAGGTCGGAC+dGTCCGACCTGGC) have been studied in solution by 1H and 13C nuclear magnetic resonance. All non-exchangeable aromatic and H-1', H-2', H-2" sugar protons were assigned using standard assignment pathways for B-DNA. All cross-peaks within these pathways were present for the perfect duplex molecule as would be expected for a right-handed A or B-form duplex. However, a few cross-peaks which would be expected in the standard case are extremely weak in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of the bulged duplex even at long mixing times (250 ms). For example, almost no cross-relaxation is observed between the H-6 proton of C22 and the H-1' of A21, directly across from the three base bulge. Yet the continuity of assignment pathways through the three base bulge argues against any discontinuous "looping out" of one or more of the extra adenosine residues. Double quantum-filtered correlated spectroscopy experiments demonstrate very little deviation from south sugar conformations for residues at or near the bulge. The perfect duplex contains three A.T basepairs as expected, resulting in three very intense T imino-AH2 cross-peaks in the H2O NOESY experiment. In contrast, only two such intense cross-peaks are observed in the same experiment using the bulged duplex sample. Assignments of the two T imino peaks using one-dimensional NOEs are consistent with disruption of the T.A base-pair immediately 3' to the bulge; this is consistent with our earlier observation of chemical reactivity at a T 3' to an An or Tn bulge. We also find evidence of disruption of the G.C base-pair immediately 5' to the bulge.

NMR study of parallel-stranded tetraplex formation by the hexadeoxynucleotide d(TG4T).

Multistranded DNA structures based upon guanine association have been proposed to be important in the structure of chromosome telomeres and in immunoglobulin class switching. Nucleic acids containing runs of guanine bases form a number of structures in vitro, including fold-back structures (Fig. 1a) and parallel-stranded quadruplex structures in DNA and RNA. The features of fold-back structures have now been determined at high-resolution. The different structures are probably based on a tetrad of hydrogen-bonded guanine bases (Fig. 1b), with buffer conditions and sequence effects mediating isomerization between the different forms. Here we use NMR spectroscopy to investigate the solution structure of the complex formed by the hexadeoxynucleotide d(TG4T) in the presence of sodium ions. We have observed the formation of a parallel-stranded quadruplex containing hydrogen-bonded tetrads of guanine. The parallel-stranded form differs significantly from the fold-back form, with individual nucleotide conformations being closer to those of B-form DNA.

Competing B-Z and helix-coil conformational transitions in supercoiled plasmid DNA.

The formation of melted regions from A + T-rich sequences and left-handed Z-DNA by alternating purine-pyrimidine sequences will both be facilitated by negative supercoiling, and thus if the sequences are present within the same plasmid molecule they will compete for the free energy of supercoiling. We have studied a series of plasmids that contain either (CG)8 or (TG)12 sequences in either G + C or A + T-rich contexts, by means of two-dimensional gel electrophoresis and chemical modification. We observe both B-Z and helix-coil transitions in all plasmids at elevated temperatures and low ionic strength. The plasmids fall into a number of different classes, in terms of the conformational behavior. As the superhelix density is increased, pCG8/vec ((CG)8 in G + C-rich context) undergoes an initial B-Z transition, followed by melting transitions in sequences remote from the (CG)8 sequence. The two transitions are coupled through the topology of the molecule but are otherwise independent. When the (CG)8 sequence was placed in an A + T-rich context (pCG8/col), the helix-coil transition was perturbed by the presence of the Z-DNA segment. Replacement of the (CG)8 tracts by (TG)12 sequences resulted in a further level of interaction between the transitions. Statistical mechanical modeling of the transitions suggested that at intermediate levels of negative supercoiling the Z-DNA formed by the (TG)12 sequence has a lowered probability due to the helix-coil transition in the A + T-rich sequences. These studies illustrate the complexities of competing conformational equilibria in supercoiled DNA molecules.

Large-scale stable opening of supercoiled DNA in response to temperature and supercoiling in (A + T)-rich regions that promote low-salt cruciform extrusion.

We have studied the properties of (A + T)-rich sequences derived from ColE1 that promote cruciform extrusion at low ionic strength in supercoiled plasmids. We compared the chemical reactivity of the sequences in negatively supercoiled DNA (using osmium tetroxide and bromoacetaldehyde) with the results of two-dimensional gel electrophoresis performed under the same conditions. Taken together, the results indicate the occurrence of cooperative helix-coil transitions in the (A + T)-rich DNA at low ionic strength, to form stable, denatured regions. The extent of the open region is a function of temperature and superhelix density, with an additional local destabilization brought about by the presence of cruciform structures. We present a simple statistical mechanical model of the helix-coil transition in the (A + T)-rich DNA, from which we have obtained estimates of the free energy for average base-pair opening of 0.31 kcal mol-1 and that for the formation of a helix-coil junction of 4.9 kcal mol-1, in 45 mM Tris-borate, pH 8.3, 0.5 mM EDTA. The results offer a model for the C-type mechanism of cruciform extrusion. Inverted repeats that are incorporated into the melted region undergo hairpin loop formation below 50 degrees C, and upon closure of the melted region, by reduction of temperature or increased ionic strength, they remain as a fully extruded cruciform structure.

B-Z cooperativity and kinetics of poly(dG-m5dC) are controlled by an unfavorable B-Z interface energy.

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m5dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl2, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl2 in 50 mM NaCl, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 +/- 0.3 kcal/mol and is length independent. Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 +/- 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event. Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.

The TFIIIA recognition fragment d(GGATGGGAG).d(CTCCCATCC) is B-form in solution.

The deoxyoligonucleotide d(GGATGGGAG).d(CTCCCATCC) is a portion of the gene recognition sequence of transcription factor IIIA (TFIIIA). The crystal structure of this oligonucleotide was shown to be A-form (Mc Call, M., Brown, T., Hunter, W.N., and Kennard, O. 1986 Nature 322, 661-664). The present study employs NMR, optical, chemical and enzymatic techniques to investigate the solution structure of this DNA 9-mer. NMR COSY experiments indicate 16 of the 18 residues are predominantly south (C2'-endo) sugar conformation. NMR NOESY indicates glycosidic angles in the range predicted for B-form DNA as opposed to A-form. Related DNA and RNA self-complementary 18-mer sequences, d(GGATGGGAGC-TCCCATCC), with U substituted for T in RNA, were studied by circular dichroism. CD spectra support B-form structures for the DNA 9-mer and the DNA 18-mer, and A-form for the RNA 18-mer. High trifluoroethanol concentrations induce a B- to A-form transition in the DNA oligonucleotides. Enzymatic and chemical probes also illustrate significant differences between the DNA and the RNA oligonucleotides. We find no evidence to support an A-form conformation for the TFIIIA recognition sequence d(GGATGGGAG).d(CTCCCATCC) in solution.