NMR studies of A.C mismatches in DNA dodecanucleotides at acidic pH. Wobble A(anti).C(anti) pair formation

Two-dimensional proton NMR studies were undertaken on the d(C-G-A-G-A-A-T-T-C-C-C-G) duplex (designated A.C 12-mer) where the A at the mismatch site is flanked by G residues and the d(C-G-C-G-A-A-T-T-C-A-C-G) duplex (designated C.A 12-mer) where the A at the mismatch site is flanked by C residues in an attempt to elucidate the role of flanking base pairs on the structure of the A.C mismatch. The exchangeable and nonexchangeable proton spectra of these two dodecanucleotides have been completely characterized by two-dimensional nuclear Overhauser enhancement (NOE) experiments in H2O and D2O solution at acidic pH. The NOE distance connectivities demonstrate that both A and C at the mismatch site are stacked into a right-handed helix between flanking G.C base pairs and exhibit anti-glycosidic torsion angles. The proton chemical shifts and NOE patterns are consistent with Wobble A.C pairing for the A.C 12-mer and C.A 12-mer duplexes in solution and demonstrate that the A.C mismatches introduce local conformational perturbations that do not extend to the central AATT segment. We detect that amino protons of adenosine (approximately 9.2 ppm) but not of cytidine at the A.C mismatch site in both duplexes on lowering the pH below 6.     

Base pair mismatches and carcinogen-modified bases in DNA: an NMR study of G.T and G.O4meT pairing in dodecanucleotide duplexes

High-resolution two-dimensional NMR studies have been completed on the self-complementary d(C-G-C-G-A-G-C-T-T-G-C-G) duplex (designated G.T 12-mer) and the self-complementary d(C-G-C-G-A-G-C-T-O4meT-G-C-G) duplex (designated G.O4meT 12-mer) containing G.T and G.O4meT pairs at identical positions four base pairs in from either end of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) spectra for the G.T 12-mer and G.O4meT 12-mer duplexes in H2O and D2O solution. The guanosine and thymidine imino protons in the G.T mismatch resonate at 10.57 and 11.98 ppm, respectively, and exhibit a strong NOE between themselves and to imino protons of flanking base pairs in the G.T 12-mer duplex. These results are consistent with wobble pairing at the G.T mismatch site involving two imino proton-carbonyl hydrogen bonds as reported previously [Hare, D. R., Shapiro, L., & Patel, D. J. (1986) Biochemistry 25, 7445-7456]. In contrast, the guanosine imino proton in the G.O4meT pair resonates at 8.67 ppm. The large upfield chemical shift of this proton relative to that of the imino proton resonance of G in the G.T mismatch or in G.C base pairs indicates that hydrogen bonding to O4meT is either very weak or absent. This guanosine imino proton has an NOE to the OCH3 group of O4meT across the pair and NOEs to the imino protons of flanking base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the stable mismatch purine-thymine in the self-complementary d(CGPuAATTTCG) duplex in solution

One- and two-dimensional nuclear Overhauser effect experiments demonstrate that a single hydrogen bond between a T imino proton and purine N3 is sufficient to hold the base pair dPu.dT in d(CGPuAATTTCG) by a Watson-Crick fashion rather than a Hoogsteen type. In addition, the dPu.dT base pair is well stacked with neighboring base pairs. The spin-lattice relaxation measurements at 30 and 35 degrees C of two decamers, d(CGPuAATTTCG) and d(CGAAATTTCG), reveal that the elimination of two single hydrogen bonds of dA.dT base pairs (due to the substitution of adenine for purine) in the sequence results in an increase in the overall imino proton exchange rate from 7 to 36 s-1 at the site of mismatch.     

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.     

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)     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

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.     

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G)

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.     

Comparison of the conformation of an oligonucleotide containing a central G-T base pair with the non-mismatch sequence by proton NMR.

We have recorded NOESY spectra of two non-selfcomplementary undecanucleotide duplexes. From the observed NOEs we do not detect any significant distortion of the helix when a G-C pair is replaced by a G-T pair and the normal interresidue connectivities can be followed through the mismatch site. We conclude that the 2D spectra of the non-exchangeable protons do not allow differentiation between a wobble or rare tautomer form for the mismatch. NOE measurements in H2O, however, clearly show that the mismatch adopts a wobble structure and give information on the hydration in the minor groove for the G-T base pair which is embedded between two A-T base pairs in the sequence.     

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site

The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.     

Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.     

Solution structure and stability of the DNA undecamer duplexes containing oxanine mismatch

Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.     

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm⁶ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm⁶ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N¹, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m⁶ A-T imino proton gives an nuclear Overhauser effect to AH² showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m₆A.T base pair by a factor of about 100.     

Pulsed FT-NMR double resonance studies of yeast tRNAPhe: specific nuclear Overhauser effects and reinterpretation of low temperature relaxation data.

Cross-relaxation effects are demonstrated between the imino protons and other protons in yeast tRNAPhe and H2O. A detailed examination has been made of the observed relaxation rate of the proton resonance at 11.8 ppm from DSS as a function of the D2O content in the solvent. This result, as well as the size and number of observed nuclear Overhauser effects, suggests that dipolar magnetization transfer between solvent H2O, amino, imino, and other tRNA protons may dominate the relaxation processes of the imino protons at low temperature. At higher temperatures the observed relaxation rate is dominated by chemical exchange. The selective nuclear Overhauser effects are shown to be an important aid in resonance assignments. By these means we were able to identify tow protons from the wobble base pair GU4 at 11.8 ppm and 10.4 ppm.     

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

One- and two-dimensional nuclear magnetic resonance (NMR) experiments have been undertaken to investigate the conformation of the d(C1-G2-C3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) self-complementary dodecanucleotide (henceforth called O6meG.C 12-mer), which contains C3.O6meG10 interactions in the interior of the helix. We observe intact base pairs at G2.C11 and G4.C9 on either side of the modification site at low temperature though these base pairs are kinetically destabilized in the O6meG.C 12-mer duplex compared to the G.C 12-mer duplex. One-dimensional nuclear Overhauser effects (NOEs) on the exchangeable imino protons demonstrate that the C3 and O6meG10 bases are stacked into the helix and act as spacers between the flanking G2.C11 and G4.C9 base pairs. The nonexchangeable base and H1', H2', H2', H3', and H4' protons have been completely assigned in the O6meG.C 12-mer duplex at 25 degrees C by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) experiments. The observed NOEs and their directionality demonstrate that the O6meG.C 12-mer is a right-handed helix in which the O6meG10 and C3 bases maintain their anti conformation about the glycosidic bond at the modification site. The NOEs between the H8 of O6meG10 and the sugar protons of O6meG10 and adjacent C9 exhibit an altered pattern indicative of a small conformational change from a regular duplex in the C9-O6meG10 step of the O6meG.C 12-mer duplex. We propose a pairing scheme for the C3.O6meG10 interaction at the modification site. Three phosphorus resonances are shifted to low field of the normal spectral dispersion in the O6meG.C 12-mer phosphorus spectrum at low temperature, indicative of an altered phosphodiester backbone at the modification site. These NMR results are compared with the corresponding parameters in the G.C 12-mer, which contains Watson-Crick base pairs at the same position in the helix.     

Sequence-specific recognition of DNA: NMR studies of the imino protons of a synthetic RNA polymerase promoter

We have synthesized both strands of a DNA duplex containing the consensus Pribnow promoter sequence TATAATG , flanked by GC base pairs to stabilize the ends of the helix. The stability of this duplex has been studied by using 1H nuclear magnetic resonance. The imino protons have been assigned by using the sequential nuclear Overhauser effect approach. Exchange rates have been monitored by using selective inversion recovery measurements. The helix is relatively unstable in the center of the AT-rich region even when surrounded by GC base pairs, and there is considerable asymmetry in the melting of the helix.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Consequences of methylation on the amino group of adenine. A proton two-dimensional NMR study of d(GGATATCC) and d(GGm6ATATCC)

A two-dimensional 500-MHz 1H-NMR study of two oligonucleotides, d(GGATATCC) and d(GGm6ATATCC), is presented in which we have investigated the effects of adenine methylation. The two-dimensional nuclear Overhauser spectra (NOESY) show that both oligonucleotides adopt a normal right-handed B-type helix and one-dimensional nuclear Overhauser enhancement (NOE) studies demonstrate that any difference in conformation must be small. However methylation drastically slows down the helix in equilibrium coil exchange which becomes slow on a proton NMR time scale. While d(GGATATCC) fits a two-site exchange model, d(GGm6ATATCC) does not and we invoke the presence of a third species which may be an intermediate in helix formation. NMR and ultraviolet spectroscopy show that methylation destabilizes the helix, measured by the melting temperature and enthalpy of dissociation.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.