Structural investigation of helices II, III, and IV of B. megaterium 5S ribosomal RNA by molecular dynamics calculations.

The structures of the helices II-III region and the helix IV region of B. megaterium 5S rRNA have been examined by means of energy minimization and molecular dynamics calculations. Calculated distances between neighboring hydrogen-bonded imino protons in helices II, III, and IV were between 3.5 and 4.5 A. The overall axis for the helices II-III region is warped rather than straight. Formation of additional Watson-Crick base pairs in loop B and loop C was not evident from the atomic positions calculated by molecular dynamics. Bases in loop C are well stacked, showing no significant change during dynamics. Bulge migration in helix III does not seem to be possible; the helices II-III region prefers one conformation. Helix II is more stable than helix III. Five base pairs in helix IV were sufficiently stable to establish that helix IV is terminated by a hairpin loop of three nucleotides. U87 protrudes from loop D. Structures of the helices II-III segment and the helix IV segment of B. megaterium 5S rRNA obtained by molecular dynamics were generally consistent with the solution structure inferred from high-field proton nmr spectroscopy.     

Identification and assignment of base pairs in three helical stems of Torulopsis utilis ribosomal 5S RNA and its RNase T1 and RNase T2 cleaved fragments via 500-MHz proton homonuclear overhauser enhancements

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.     

NMR studies of an exocyclic 1,N2-propanodeoxyguanosine adduct (X) located opposite deoxyadenosine (A) in DNA duplexes at basic pH: simultaneous partial intercalation of X and A between stacked bases

The NMR parameters for the 1,N2-propanodeoxyguanosine (X) opposite deoxyadenosine positioned in the center of the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) X.A 9-mer duplex are pH dependent. A previous paper established protonated X5(syn).A14(anti) pairing in the X.A 9-mer duplex at pH 5.8 [Kouchakdjian, M., Marinelli, E., Gao, X., Johnson, F., Grollman, A., & Patel, D. J. (1989) Biochemistry 28, 5647-5657]; this paper focuses on the pairing alignment at the lesion site at pH 8.9. The observed NOEs between specific exocyclic CH2 protons and both the imino proton of G6 and the sugar H1' protons of C13 and A14 establish that X5 is positioned toward the G6.C13 base pair with the exocyclic ring directed between C13 and A14 on the partner strand. The observed NOE between the H2 proton of A14 and the imino proton of G4, but not G6, establishes that A14 at the lesion site is directed toward the G4.C15 base pair. NOEs are detected between all exocyclic CH2 protons of X5 and the H2 proton of A14, confirming that both X5 and A14 are directed toward the interior of the helix. The X5(anti).A14(anti) alignment at pH 8.9 is accommodated within the helix with retention of Watson-Crick pairing at flanking G4.C15 and G6.C13 base pairs. The energy-minimized conformation of the (G4-X5-G6).(C13-A14-C15) segment at pH 8.9 establishes that X5 and A14 are directed into the helix, partially stack on each other, and are not stabilized by intermolecular hydrogen bonds. The X5 base is partially intercalated between C13 and A14 on the unmodified strand, while A14 is partially intercalated between G4 and X5 on the modified strand. This results in a larger separation between the G4.C15 and G6.C13 base pairs flanking the lesion site in the basic pH conformation of the X.A 9-mer duplex. The midpoint of the transition between the protonated X5(syn).A14(anti) and X5(anti).A14(anti) conformations occurs at pH 7.6, establishing an unusually high pKa for protonation of the A14 ring opposite the X5 exocyclic adduct site. Thus, the interplay between hydrophobic and hydrogen-bonding contributions modulated by pH defines the alignment of 1,N2-propanodeoxyguanosine opposite deoxyadenosine in the interior of DNA helices.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes

High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes.     

Identification and assignment of base pairs in the "tuned helix" of intact and ribonuclease T1 cleavage fragments of wheat germ ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements

Wheat germ has been chosen as a representative eukaryote for study of ribosomal 5S RNA secondary structure. Proton homonuclear Overhauser enhancements (NOE's) at 500 MHz for the hydrogen-bonded base-pair protons in the 10-15 ppm region are used to establish the identity (A X U, G X C, or G X U) and base-pair sequence (e.g., G X C-A X U-C X G) within a given helical segment. Assignment of that segment to particular base pairs in the secondary structure is based upon NOE's conducted at different temperatures (to determine which signals "melt" together), variation of salt conditions (to produce differential chemical shifts in order to better distinguish components of an unresolved spectral envelope), and isolation and purification of RNase T1 cleavage fragments (in order to reduce the spectrum to just a few base pairs). The NOE patterns for the RNase T1 fragments are the same as in the intact 5S RNA, supporting the assumption that structural features of this region in the intact 5S RNA are preserved in the fragment. Chemical shifts predicted from ring current induced effects for a proposed base-pair sequence are then compared to experimental chemical shifts. By these methods, a portion of the "tuned helix" segment (namely, the base-pair sequence C18G60-A19U59-C20G58) is demonstrated spectroscopically for the first time in any 5S RNA. The tuned helix and common arm segments are less stable than the rest of the molecule. Variation of sodium and magnesium levels reveals multiple configurations of the wheat germ 5S RNA in solution.     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of exocyclic 1,N2-propanodeoxyguanosine adducts (X) opposite purines in DNA duplexes: protonated X(syn).A(anti) pairing (acidic pH) and X(syn).G(anti) pairing (neutral pH) at the lesion site

Proton and phosphorus two-dimensional NMR studies are reported for the complementary d(C1-A2-T3-G4-X5-G6-T7-A8-C9).d(G10-T11-A12-C13-A14-C15-A 16-T17-G18) nonanucleotide duplex (designated X.A 9-mer) that contains a 1,N2-propanodeoxyguanosine exocyclic adduct, X5, opposite deoxyadenosine A14 in the center of the helix. The NMR studies detect a pH-dependent conformational transition; this paper focuses on the structure present at pH 5.8. The two-dimensional NOESY studies of the X.A 9-mer duplex in H2O and D2O solution establish that X5 adopts a syn orientation while A14 adopts an anti orientation about the glycosidic bond at the lesion site. The large downfield shift of the amino protons of A14 demonstrates protonation of the deoxyadenosine base at pH 5.8 such that the protonated X5(syn).A14(anti) pair is stabilized by two hydrogen bonds at low pH. At pH 5.8, the observed NOE between the H8 proton of X5 and the H2 proton of A14 in the X.A 9-mer duplex demonstrates unequivocally the formation of the protonated X5(syn).A14(anti) pair. The 1,N2-propano bridge of X5(syn) is located in the major groove. Selective NOEs from the exocyclic methylene protons of X5 to the major groove H8 proton of flanking G4 but not G6 of the G4-X5-G6 segment provide additional structural constraints on the local conformation at the lesion site. A perturbation in the phosphodiester backbone is detected at the C13-A14 phosphorus located at the lesion site by 31P NMR spectroscopy. The two-dimensional NMR studies have been extended to the related complementary X.G 9-mer duplex that contains a central X5.G14 lesion in a sequence that is otherwise identical with the X.A 9-mer duplex. The NMR experimental parameters are consistent with formation of a pH-independent X5(syn).G14(anti) pair stabilized by two hydrogen bonds with the 1,N2-propano exocyclic adduct of X5(syn) located in the major groove.     

Proton exchange and internal motions in two chromomycin dimer-DNA oligomer complexes.

Previous structural studies on the complexes of the chromomycin (CHR) dimer with duplexes of d(A1-A2-G3-G4-C5-C6-T7-T8) and of d(A1-G2-G3-A4-T5-C6-C7-T8) in solution [one Mg(II) and two drugs per duplex] are extended to hydrogen exchange measurements. Exchange of the OH8 proton of chromomycin, measured by real time proton-deuterium exchange, is very slow and requires dissociation of the complex, whose lifetime is thus determined. The lifetimes and apparent dissociation constants of base pairs are deduced from the catalysis of imino proton exchange by ammonia. The four central base pairs, which interact with the CHR chromophores in the minor groove (Gao & Patel, 1990), may open within the complex, but the opening rate is less than in the free duplex by one to two orders of magnitude. The activation energy for base-pair opening and the differences between the lifetimes of adjacent pairs suggest that single base-pair opening is the predominant imino proton exchange pathway in all cases. In the symmetrical complex of chromomycin with the first duplex, the lifetimes of the central base pairs (G3.C6 and G4.C5) are in the same range (52 and 29 ms, respectively, at 38 degrees C). In the asymmetrical complex formed with the second duplex, the base-pair lifetimes in the G2-G3-A4-T5 segment that interacts with the chromophore moiety are strongly increased. That of G3.C6 is particularly long. Above 50 degrees C, exchange of the G3 imino proton is opening limited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of three G.U base pairs in Bacillus subtilis ribosomal 5S RNA via 500-MHz proton homonuclear Overhauser enhancements

Three distinct G.U base pairs in Bacillus subtilis 5S RNA have been identified via homonuclear Overhauser enhancements (NOE) of their low-field (9-15 ppm) proton Fourier transform nuclear magnetic resonances at 11.75 T. With these G.U resonances as starting points, short segments of NOE connectivity can be established. One G.U-G.C-G.C segment (most probably G4.C112-G5.C111-U6.G110) can definitely be assigned to the terminal helix. The existence of at least part of the terminal helical stem of the secondary structure of a Gram-positive bacterial 5S RNA has thus been established for the first time by direct experimental observation. Addition of Mg2+ produces almost no conformational changes in the terminal stem but results in major conformational changes elsewhere in the structure, as reflected by changes in the 1H 500-MHz low-field NMR spectrum. Assignment of the two remaining G.U base pairs will require further experiments (e.g., enzymatic-cleavage fragments). Finally, the implications of these results for analysis of RNA secondary structure are discussed.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

Solution conformation of the (+)-trans-anti-benzo[g]chrysene-dA adduct opposite dT in a DNA duplex.

The solution structure of the adduct derived from the covalent bonding of the fjord region (+)-(11S, 12R, 13R, 14S) stereoisomer of anti -11,12-dihydroxy-13,14-epoxy-11,12,13, 14-tetrahydrobenzo[g]chrysene, (+)- anti -B[g]CDE, to the exocyclic N(6)amino group of the adenine residue dA6, (designated (+)- trans-anti -(B[g]C)dA6), positioned opposite a thymine residue dT17 in the DNA sequence context d(C1-T2-C3-T4-C5-(B[g]C)A6-C7-T8-T9-C10-C11). d(G12-G13-A14-A15-G16-T17-G18-A19-G20++ +-A21-G22) (designated (B[g]C)dA. dT 11-mer duplex), has been studied using structural information derived from NMR data in combination with molecular dynamics (MD) calculations. The solution structure of the (+)- trans-anti -(B[g]C)dA.dT 11-mer duplex has been determined using an MD protocol where both interproton distance and dihedral angle restraints deduced from NOESY and COSY spectra are used during the refinement process, followed by additional relaxation matrix refinement to the observed NOESY intensities to account for spin diffusion effects. The results established that the covalently attached benzo[g]chrysene ring intercalates into the DNA helix directed towards the 5'-side of the modified strand and stacks predominantly with dT17 when intercalated between dC5.dG18 and (B[g]C)dA6.dT17 base-pairs. All base-pairs, including the modified (B[g]C)dA6.dT17 base-pair, are aligned through Watson-Crick pairing as in normal B -DNA. In addition, the potential strain associated with the highly sterically hindered fjord region of the aromatic portion of the benzo[g]chrysenyl ring is relieved through the adoption of a non-planar, propeller-like geometry within the chrysenyl ring system. This conformation shares common structural features with the related (+)- trans-anti -(B[c]Ph)dA adduct in the identical base sequence context, derived from the fjord region (+)-(1S,2R,3R,4S)-3, 4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene stereoisomer, in which intercalation is also observed towards the 5'-side of the modified dA6.dT17 base-pair.     

The contribution of pseudouridine to stabilities and structure of RNAs

Thermodynamic data are reported revealing that pseudouridine (Ψ) can stabilize RNA duplexes when replacing U and forming Ψ-A, Ψ-G, Ψ-U and Ψ-C pairs. Stabilization is dependent on type of base pair, position of Ψ within the RNA duplex, and type and orientation of adjacent Watson–Crick pairs. NMR spectra demonstrate that for internal Ψ-A, Ψ-G and Ψ-U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. Ψ has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow Ψ to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of Ψ.     

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling.

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.     

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].A[anti] pair formation and its pH dependence

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)-induced hydrolysis.

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)-induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single-stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA-treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33-C42 and G79-C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)-induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)-hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.     

Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]G.C adduct structure and comparison with the (+)-trans-anti-[BP]G.C enantiomer.

Benzo[a]pyrene (BP) is an environmental genotoxin, which, following metabolic activation to 7,8-diol 9,10-epoxide (BPDE) derivatives, forms covalent adducts with cellular DNA. A major fraction of adducts are derived from the binding of N2 of guanine to the C10 position of BPDE. The mutagenic and carcinogenic potentials of these adducts are strongly dependent on the chirality at the four asymmetric benzylic carbon atoms. We report below on the combined NMR-energy minimization refinement characterization of the solution conformation of (-)-trans-anti-[BP]G positioned opposite C and flanked by G.C base pairs in the d(C1-C2-A3-T4-C5-[BP]G6-C7-T8-A9-C10-C11).d(G12-G13-T14++ +-A15-G16-C17- G18-A19-T20-G21-G22) duplex. Two-dimensional NMR techniques were applied to assign the exchangeable and non-exchangeable protons of the benzo[a]pyrenyl moiety and the nucleic acid in the modified duplex. These results establish Watson-Crick base pair alignment at the [BP]G6.C17 modification site, as well as the flanking C5.G18 and C7.G16 pairs within a regular right-handed helix. The solution structure of the (-)-trans-anti-[BP]G.C 11-mer duplex has been determined by incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOE buildup curves as constraints in energy minimization computations. The BP ring spans both strands of the duplex in the minor groove and is directed toward the 3'-end of the modified strand in the refined structure. One face of the BP ring of [BP]G6 stacks over the C17 residue across from it on the partner strand while the other face is exposed to solvent.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.