Isolating the roles of different forcing agents in global stratospheric temperature changes using model integrations with incrementally added single forcings.

Satellite instruments show a cooling of global stratospheric temperatures over the whole data record (1979-2014). This cooling is not linear, and includes two descending steps in the early 1980s and mid-1990s. The 1979-1995 period is characterized by increasing concentrations of ozone depleting substances (ODS) and by the two major volcanic eruptions of El Chichón (1982) and Mount Pinatubo (1991). The 1995-present period is characterized by decreasing ODS concentrations and by the absence of major volcanic eruptions. Greenhouse gas (GHG) concentrations increase over the whole time period. In order to isolate the roles of different forcing agents in the global stratospheric temperature changes, we performed a set of AMIP-style simulations using the NASA Goddard Earth Observing System Chemistry-Climate Model (GEOSCCM). We find that in our model simulations the cooling of the stratosphere from 1979 to present is mostly driven by changes in GHG concentrations in the middle and upper stratosphere and by GHG and ODS changes in the lower stratosphere. While the cooling trend caused by increasing GHGs is roughly constant over the satellite era, changing ODS concentrations cause a significant stratospheric cooling only up to the mid-1990s, when they start to decrease because of the implementation of the Montreal Protocol. Sporadic volcanic events and the solar cycle have a distinct signature in the time series of stratospheric temperature anomalies but do not play a statistically significant role in the long-term trends from 1979 to 2014. Several factors combine to produce the step-like behavior in the stratospheric temperatures: in the lower stratosphere, the flattening starting in the mid 1990's is due to the decrease in ozone depleting substances; Mount Pinatubo and the solar cycle cause the abrupt steps through the aerosol-associated warming and the volcanically induced ozone depletion. In the middle and upper stratosphere, changes in solar irradiance are largely responsible for the step-like behavior of global temperatures anomalies, together with volcanically induced ozone depletion and water vapor increases in the post-Pinatubo years.

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG.

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

Stopped-flow and Brownian dynamics studies of electrostatic effects in the kinetics of binding of 7-methyl-GpppG to the protein eIF4E.

The kinetics of binding 7-methyl-GpppG, an analogue of the 5'-mRNA cap, to the cap-binding protein eIF4E, at 20 degrees C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic strengths the ligand must be closer to the binding site to induce the fluorescence quenching.

Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues.

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.

Multiple isoforms of eukaryotic protein synthesis initiation factor 4E in Caenorhabditis elegans can distinguish between mono- and trimethylated mRNA cap structures.

The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2, 7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.

Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic N4-hydroxycytosine.

UV and NMR spectroscopy were employed to study the tautomerism, acid-base properties and conformation of the exocyclic N(4)-OH group in 1-methyl-N(4)-hydroxycytosine (1-mOH(4)C), and its methyl derivatives, viz. the fixed imino forms (1,3-m(2)OH(4)C and 1,3,5-m(3)OH(4)C), the fixed amino form (1,N(4)-m(2)OH(4)C), and analogues sterically constrained to the form syn (1,5-m(2)OH(4)C) or anti (1,3-m(2)OH(4)C) with respect to the ring N(3). Relative to 1,N(4)-m(2)OH(4)C, UV spectroscopy showed that the other analogues were predominantly imino and that all analogues formed a structurally common cation in acid medium, with results pointing to approximately 90% population of the imino species for 1-mOH(4)C and 1,5-m(2)OH(4)C, further supported by NMR spectroscopy. Both exhibited two sequential dissociations in alkaline medium, the first due to N(4)-OH, followed by the N(3)-H. (1)H and (13)C NMR spectroscopy showed 1-mOH(4)C in the conformation syn. With 1,3,5-m(3)OH(4)C, an ;overcrowded' planar molecule with steric constraints to both the syn and anti conformations, a syn-anti equilibrium is observed, with a preference of approximately 75% for the anti rotamer, independently of the polarity of the medium. Exchange between the rotamers is slow on the NMR time-scale, with a minimal barrier to exchange exceeding 100 kJ/mol. In low-polar media, the analogues associate as dimers via O(4)-Hcdots, three dots, centeredO(2) or O(4)-Hcdots, three dots, centeredN(4) hydrogen bonds, with association constants at ambient temperature of 4.6 (1,3-m(2)OH(4)C), 12.8 (anti 1,3,5-m(3)OH(4)C), 36 (1,5-m(2)OH(4)C), 109 (syn 1,3,5-m(3)OH(4)C) M(-1). Implications of the overall findings to the promutagenic activities of OH(4)C and OMe(4)C are examined.

Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues.

Intramolecular stacking of a series of new synthesized dinucleotide mRNA cap analogues has been investigated in aqueous buffers by means of fluorescence and 1H-NMR at various pH and temperatures, and compared with that for 7-methylguanosine(5')ppp(5')guanosine (m7GpppG), as well as its hypermethylated derivative m(3)2,2,7GpppG. Thermodynamic parameters for intramolecular self-association stabilized by stacking were established by temperature-dependent fluorescence quenching, taking into account collisional deactivation of the excited states. Relative orientations of the stacked bases in the cap analogues were determined with the aid of a program GEOSHIFT (Stolarski et al., Biochim. Biophys. Acta (1996) 1293, 97), based on ring-current anisotropy. 1D-soft-TOCSY experiments were applied to extract the exact values of vicinal coupling constants, and hence to resolve solution conformation of the cap molecules. Stacking interaction has been discussed in detail in terms of the cap structural features, e.g., types of bases and length of the 5',5'-phosphate bridges, and regarding the interactions stabilizing intramolecular stacking.

1H-NMR studies on association of mRNA cap-analogues with tryptophan-containing peptides.

1H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2-8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m7GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m7G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 angstroms apart. The structures of the stacked complexes were determined. In particular, stacking between m(2,2,7)3G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m7G and m(2,7)2G and indole. With the dinucleotide cap-analogues, only the m7G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m7G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring N1H (pKa approximately 7.3).

Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209.

Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.

Structure and conformation of the cyclic phosphate of Ganciclovir, a broad-spectrum antiviral agent.

The title compound, the cyclic phosphate of the antiviral acyclonucleoside Ganciclovir (2'-NDG, DHPG), is itself a potent broad-spectrum antiviral agent, but with a different mechanism of action. The cyclic phosphate, 9-[[[(2-hydroxy-1,3,2-dioxophosphorinan-5-yl)oxy]methyl]-P- oxide]guanine (2'-nor-cGMP,DHPG-cMP), crystallizes in the monoclinic space group P2(1)/n with unit cell dimensions a = 6.612(1) A, b = 11.562(4) A, c = 19.231(5) A and beta = 91.786(2) degrees at -165 degrees C. The N7 of the guanine base is protonated, so that the molecule is in a zwitterionic form, with two water molecules in the asymmetric unit. The principal conformational features of DHPG-cMP in the crystal are as follows: the acyclic chain is partially folded; the six-membered cyclic phosphate ring is in a chair form with C3', O3', C5' and O5' in a plane; P and C4' are displaced in diametrically opposite directions from this plane; the O4' is in the axial orientation with respect to this ring; and the aglycon is in the high syn conformation about the glycosidic bond. The conformation of the cyclic phosphate ring in aqueous medium, determined by means of 1H-NMR spectroscopy, is similar to that in the crystalline form. The conformational features of DHPG-cMP were compared with those of the parent DHPG and other related compounds and, in particular, with those of the second messenger 3':5'-cGMP, of which it is a close structural analogue. Previously reported substrate/inhibitor properties of these compounds in several enzyme systems are examined in relation to the possible mechanism of antiviral activity of DHPG-cMP as a second messenger analogue of cGMP.

Record low global ozone in 1992.

The 1992 global average total ozone, measured by the Total Ozone Mapping Spectrometer (TOMS) on the Nimbus-7 satellite, was 2 to 3 percent lower than any earlier year observed by TOMS (1979 to 1991). Ozone amounts were low in a wide range of latitudes in both the Northern and Southern hemispheres, and the largest decreases were in the regions from 10 degrees S to 20 degrees S and 100N to 60 degrees N. Global ozone in 1992 is at least 1.5 percent lower than would be predicted by a statistical model that includes a linear trend and accounts for solar cycle variation and the quasi-biennial oscillation. These results are confirmed by comparisons with data from other ozone monitoring instruments: the SBUV/2 instrument on the NOAA-11 satellite, the TOMS instrument on the Russian Meteor-3 satellite, the World Standard Dobson Instrument 83, and a collection of 22 ground-based Dobson instruments.

Cyclic phosphates of some antiviral acyclonucleosides: relationship between conformation and substrate/inhibitor properties in some enzyme systems.

Solution conformations, and substrate/inhibitor properties towards several phosphodiesterases and other nucleolytic enzymes, have been investigated for the cyclic phosphates of various acyclonucleosides, some with known antiviral activity, including 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG) and its carbocyclic congener, 9-(3,4-dihydroxybutyl)guanine (DHBG),9-[4-hydroxy-2-(hydroxymethyl)- butyl]-guanine (2HM-HBG), and 9-(2,3-dihydroxypropyl)guanine (HPG). Conformations of the cyclic phosphate rings were derived from analysis of the 1H-1H and 1H-31P vicinal coupling constants in the 1H n.m.r. spectra, subsequently optimalized by minimalization of the internal energy. The resulting structures were examined with respect to their ability to recognize various specific phosphodiesterases and nucleases, and some structural parameters were delineated for acyclonucleotide interactions. Qualitative data are presented for inhibitory properties of the acyclonucleoside cyclic phosphates, and, in those instances where they were substrates, kinetic constants were evaluated. An unusual finding was the apparent ability of nuclease P1 to hydrolyse a five-membered cyclic phosphate ring of an acyclonucleoside.

Solution structure of the EcoRI DNA octamer containing 5-fluorouracil via restrained molecular dynamics using distance and torsion angle constraints extracted from NMR spectral simulations.

The self-complementary DNA octamer [d(GGAATUFCC)]2, containing the EcoRI recognition sequence with one of the thymines replaced by 5-fluorouracil (UF), was synthesized. Proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra, as well as one-dimensional spectra at different temperatures, were recorded for the octamer. Consequently, all proton resonances were assigned. The thermally induced transition from the duplex to single strands has been followed, demonstrating the stability of the duplex containing 5-fluorouracil. Simulations of the 2QF-COSY cross-peaks by means of the programs SPHINX and LINSHA were compared with experimental data, establishing scalar coupling constants for the sugar ring protons and hence sugar pucker parameters. The deoxyribose rings exhibit a dynamic equilibrium of N- and S-type conformers with 75-95% populations of the latter. Two programs used for complete relaxation matrix analysis 2D NOE spectra, CORMA and MARDIGRAS, were modified to account for the influence of the fluorines on dipolar interactions in the proton system. Quantitative assessment of the 2D NOE cross-peak intensities for different mixing times, in conjunction with the program MARDIGRAS, gave a set of interproton distances for each mixing time. The largest and smallest values of each of the interproton distances were chosen as the upper and lower bounds for each distance constraint. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 35 ps were performed, utilizing 284 experimental distance and torsion angle constraints and two different starting structures, energy-minimized A- and B-DNA. Convergence to similar structures with a root-mean-square deviation of 1.2 A was achieved for the central hexamer of the octamer, starting from A- and B-DNA. The average structure from six different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and showed a substantial improvement of the 2D NOE sixth-root residual index in comparison with classical and energy-minimized B-DNA. A detailed analysis of the conformation of the final structure and comparison with structures of similar sequences, obtained by different methods, were performed.

Measured trends in stratospheric ozone.

Recent findings, based on both ground-based and satellite measurements, have established that there has been an apparent downward trend in the total column amount of ozone over mid-latitude areas of the Northern Hemisphere in all seasons. Measurements of the altitude profile of the change in the ozone concentration have established that decreases are taking place in the lower stratosphere in the region of highest ozone concentration. Analysis of updated ozone records, through March of 1991, including 29 stations in the former Soviet Union, and analysis of independently calibrated satellite data records from the Total Ozone Mapping Spectrometer and Stratospheric Aerosol and Gas Experiment instruments confirm many of the findings originally derived from the Dobson record concerning northern midlatitude changes in ozone. The data from many instruments now provide a fairly consistent picture of the change that has occurred in stratospheric ozone levels.

Monophosphates and cyclic phosphates of some antiviral acyclonucleosides: synthesis, conformation and substrate/inhibitor properties in some enzyme systems.

Chemical and enzymatic procedures are described for the synthesis of the monophosphates and cyclic phosphates of the antiviral acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG), its 3-hydroxymethyl-4-hydroxybutyl analogue, the (R)- and (S)-epimers of 9-(3,4-dihydroxybutyl)guanine, and 9-(2,3-dihydroxypropyl)guanine. The structures, and some conformational features, of all the foregoing, were determined by 1H and 31P NMR spectroscopy. Their substrate/inhibitor properties have been examined in several enzyme systems, including ribonucleases, snake venom phosphodiesterase, beef heart and higher plant cyclic nucleotide phosphodiesterases, nuclease P1, and 3'- and 5'-nucleotidases. The enzymatic results are considered in relation to the mechanism of the antiviral activity of the cyclic phosphate of DHPG.

Solution conformations of some acyclo nucleoside and nucleotide analogues of antiviral acyclonucleosides, and their substrate/inhibitor properties in several enzyme systems.

Chemical and enzymatic procedures have been employed for the preparation of various phosphorylated derivatives of the acyclonucleoside 9-(1,3-dihydroxy-2-propoxymethyl)adenine, an analogue of the active antiviral agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG). In combination with the previously reported 2',3'-seco nucleosides and their phosphates and cyclic phosphates (Stolarski et al., Z. Naturforsch. 41c, 758-770, 1986), this made available a broad class of acyclonucleosides and nucleotides, the acyclic moieties of which are capable of mimicking the ribose and 2'-deoxyribose rings. The solution conformations of the foregoing were determined with the aid of 1H, 13C and 31P NMR, and compared with those of DHPG and 9-(hydroxyethoxymethyl)guanine (Acyclovir, ACV). Particular attention was devoted to conformations about C-O bonds in different acyclic fragments, which demonstrated well-defined differences between 2',3'-seco derivatives on the one hand (conformational "rigidity") and derivatives with DHP and AC acyclic chains on the other (rotation about the C(1')-O(4') bond). The overall results are in good general agreement with reported crystal structures, and are compared with those obtained by quantum mechanical calculations. The conformational features of the various compounds are also discussed in relation to their substrate and/or inhibitor properties in a number of enzyme systems, including adenosine deaminase, phosphodiesterases, nuclease P1,3'-nucleotidase and herpes virus type 1 thymidine kinase.

One- and two-dimensional 1H-NMR investigations of two 19-base-pair analogues of the tet operator.

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.

Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine.

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.