Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA

19F nuclear magnetic resonance has been used to study fully active Escherichia coli tRNA1Val in which 5-fluorouracil has replaced more than 90% of all uracil and uracil-derived modified bases. The 19F spectrum of the native tRNA contains resolved resonances for all 14 incorporated 5-fluorouracils. These are spread over a 6 ppm range, from 1.8 to 7.7 ppm downfield of the standard free 5-fluorouracil. The 19F resonances serve as sensitive monitors of tRNA conformation. Removal of magnesium or addition of NaCl produces major, reversible changes in the 19F spectrum. Most affected is the lowest field resonance (peak A) in the spectrum of the native tRNA. This shifts 2-3 ppm upfield as the Mg2+ concentration is lowered or the NaCl concentration is raised. Thermal denaturation of the tRNA results in a collapse of the spectrum to a single broad peak centered at 4.7 ppm. Study of the pH dependence of the 19F spectrum shows that five incorporated fluorouracils with 19F signals in the central, 4-5.5 ppm, region of the spectrum, peaks C, D, E, F, and H, are accessible to titration in the pH 4.5-9 range. All have pKa's close to that of free 5-fluorouridine (ca. 7.5). Evidence for a conformation change in the tRNA at mildly acidic pHs, ca. 5.5, is also presented. Four of the titratable 5-fluorouracil residues, those corresponding to peaks D, E/F, and H in the 19F spectrum of fluorine-labeled tRNAVal1, are essentially completely exposed to solvent as determined by the solvent isotope shift (SIS) on transfer of the tRNA from H2O to 2H2O. These are also the 5-fluorouracils that readily form adducts with bisulfite, a reagent that reacts preferentially with pyrimidines in single-stranded regions. On the basis of these results, resonances D, E, F, and H in the middle of the 19F spectrum are attributed to 5-fluorouracils in non-base-paired (loop) regions of the tRNA. Evidence from the ionic strength dependence of the 19F spectrum and arguments based on other recent studies with fluorinated tRNAs support earlier suggestions [Horowitz, J., Ofengand, J., Daniel, W. E., & Cohn, M. (1977) J. Biol. Chem. 252, 4418-4420] that the resonances at lowest field correspond to tertiary hydrogen-bonded 5-fluorouracils. Consideration of ring-current effects and the preferential perturbation of upfield 19F resonances by the cyclophotoaddition of 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, which is known to react most readily with pyrimidines in double-stranded regions, permits initial assignment of upfield resonances to 5-fluorouracils in helical stems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorine-19 nuclear magnetic resonance as a probe of the solution structure of mutants of 5-fluorouracil-substituted Escherichia coli valine tRNA.

In order to utilize 19F nuclear magnetic resonance (NMR) to probe the solution structure of Escherichia coli tRNAVal labeled by incorporation of 5-fluorouracil, we have assigned its 19F spectrum. We describe here assignments made by examining the spectra of a series of tRNAVal mutants with nucleotide substitutions for individual 5-fluorouracil residues. The result of base replacements on the structure and function of the tRNA are also characterized. Mutants were prepared by oligonucleotide-directed mutagenesis of a cloned tRNAVal gene, and the tRNAs transcribed in vitro by bacteriophage T7 RNA polymerase. By identifying the missing peak in the 19F NMR spectrum of each tRNA variant we were able to assign resonances from fluorouracil residues in loop and stem regions of the tRNA. As a result of the assignment of FU33, FU34 and FU29, temperature-dependent spectral shifts could be attributed to changes in anticodon loop and stem conformation. Observation of a magnesium ion-dependent splitting of the resonance assigned to FU64 suggested that the T-arm of tRNAVal can exist in two conformations in slow exchange on the NMR time scale. Replacement of most 5-fluorouracil residues in loops and stems had little effect on the structure of tRNAVal; few shifts in the 19F NMR spectrum of the mutant tRNAs were noted. However, replacing the FU29.A41 base-pair in the anticodon stem with C29.G41 induced conformational changes in the anticodon loop as well as in the P-10 loop. Effects of nucleotide substitution on aminoacylation were determined by comparing the Vmax and Km values of tRNAVal mutants with those of the wild-type tRNA. Nucleotide substitution at the 3' end of the anticodon (position 36) reduced the aminoacylation efficiency (Vmax/Km) of tRNAVal by three orders of magnitude. Base replacement at the 5' end of the anticodon (position 34) had only a small negative effect on the aminoacylation efficiency. Substitution of the FU29.A41 base-pair increased the Km value 20-fold, while Vmax remained almost unchanged. The FU4.A69 base-pair in the acceptor stem, could readily be replaced with little effect on the aminoacylation efficiency of E. coli tRNAVal, indicating that this base-pair is not an identity element of the tRNA, as suggested by others.     

Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA. A 19F nuclear magnetic resonance relaxation study

19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion. Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz. Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2. The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz. Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule. The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms. Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside. These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns. By using available 19F n.m.r. assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue. Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees). A correlation between residue mobility and solvent exposure is also demonstrated. The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).     

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.     

Fluorine-19 NMR studies of the thermal unfolding of 5-fluorouracil-substituted Escherichia coli valine transfer RNA.

19F NMR spectroscopy was used to monitor the thermal unfolding of E. coli tRNAVal labeled by incorporation of 5-fluorouracil (FUra). With rising temperatures, resonances in the 19F NMR spectrum of (FUra)tRNAVal gradually shift towards the central region of the spectrum and merge into a single broad peak above 85 degrees C. FU55 and FU12 are the first to shift, beginning at temperatures below 40 degrees C, which suggests that the initial steps of thermal denaturation of tRNAVal involve disruption of the tertiary interactions between the D- and T-arms. The acceptor stem and the FU64-G50 wobble base pair in the T-stem are particularly stable to thermal denaturation. A temperature-dependent splitting of the 19F resonance assigned to FU64, at temperatures above 40 degrees C, suggests that the T-arm of (FUra)tRNAVal exists in two conformations in slow exchange on the NMR time scale.     

Fluorine-19 nuclear magnetic resonance investigation of fluorine-19-labeled phospholipids. 1. A multiple-pulse study

A multiple-pulse nuclear magnetic resonance technique has been used to measure the order parameter, SFF, at 40 MHz for dimyristoylphosphatidylcholine labeled with a difluoromethylene group at the 4-, 8-, or 12-position of the sn-2-acyl chain dispersed in water in the liquid-crystalline phase. The Carr-Purcell-Meiboom-Gill multiple-pulse sequence can resolve the homonuclear dipolar coupling between the two fluorine nuclei, thus making a direct determination of the order parameter, SFF, for the F-F internuclear vector possible. Other interactions, such as the 19F chemical shift anisotropy, heteronuclear dipolar couplings, and field inhomogeneity, which normally obscure the dipolar splitting, are effectively canceled. The order parameters obtained in this work compare well with those obtained by 19F nuclear magnetic resonance line-shape analysis of the 19F-labeled phospholipids reported in the following paper [Dowd, S. R., Simplaceanu, V., & Ho, C. (1984) Biochemistry (following paper in this issue)] as well as comparable SCD order parameters, determined for the deuterium-carbon internuclear vector of deuterium-labeled phospholipids [Oldfield, E., Meadows, M., Rice, D., & Jacobs, R. (1978) Biochemistry 17, 2727-2740]. The present results clearly show the usefulness of using nuclear magnetic resonance spectroscopy to investigate lipid-lipid and protein-lipid interactions, especially for those systems containing a difluoromethylene group in the acyl chain of a phospholipid molecule.     

Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.     

Fluorine-19 nuclear magnetic resonance studies of lipid phase transitions in model and biological membranes.

Fluorinated fatty acids of the general formula CH3(CH2)13-mCF2(CH2)m-2COOH are informative spectroscopic probes of the gel to liquid-crystalline phase transitions in phospholipid dispersions and in biological membranes. We present theoretical considerations to suggest that the 19F nuclear magnetic resonance line shapes are very different for frozen and fluid lipid regions. Our studies confirm this expectation for mixed phospholipid multilamellar dispersions containing a trace of difluoromyristate. The method correctly measures the onset and completion temperatures of the transition in the well-studied dimyristoylphosphaditylcholine distearoylphosphatidylcholine system and also describes the motional behavior of the solid and fluid phases within the transition. Lipids extracted from Escherichia coli membranes show similar motional phenomena through the transition-temperature range according to 19F nuclear magnetic resonance studies of difluoromyristate biosynthetically incorporated into the K1060B5 strain, an unsaturated fatty acid auxotroph. Intact cells or membrane vesicles show substantially different behavior from extracted lipids, indicating that membrane proteins significantly perturb the phase transition. Evidence presented in this paper also shows that the 19F resonance from Escherichia coli phospholipids is sensitive to various intramembrane interactions. There is a general decrease in restriction of motion due to neutral lipids and an opposite effect due to the architecture of the native membrane. Neither effect is temperature sensitive. However, there are interactions in the intact membrane, affecting the 19F resonance, that are temperature dependent both due to the phase-transition process and due to processes occurring at high temperatures.     

Fluorine-19 magnetic resonance angiography of the mouse

Purpose

To implement and characterize a fluorine-19 (¹⁹F) magnetic resonance imaging (MRI) technique and to test the hypothesis that the ¹⁹F MRI signal in steady state after intravenous injection of a perfluoro-15-crown-5 ether (PCE) emulsion may be exploited for angiography in a pre-clinical in vivo animal study.

Materials and Methods

In vitro at 9.4T, the detection limit of the PCE emulsion at a scan time of 10 min/slice was determined, after which the T₁ and T₂ of PCE in venous blood were measured. Permission from the local animal use committee was obtained for all animal experiments. 12 µl/g of PCE emulsion was intravenously injected in 11 mice. Gradient echo ¹H and ¹⁹F images were obtained at identical anatomical levels. Signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were determined for 33 vessels in both the ¹⁹F and ¹H images, which was followed by vessel tracking to determine the vessel conspicuity for both modalities.

Results

In vitro, the detection limit was ∼400 µM, while the ¹⁹F T₁ and T₂ were 1350±40 and 25±2 ms. The ¹⁹F MR angiograms selectively visualized the vasculature (and the liver parenchyma over time) while precisely coregistering with the ¹H images. Due to the lower SNR of ¹⁹F compared to ¹H (17±8 vs. 83±49, p<0.001), the ¹⁹F CNR was also lower at 15±8 vs. 52±35 (p<0.001). Vessel tracking demonstrated a significantly higher vessel sharpness in the ¹⁹F images (66±11 vs. 56±12, p = 0.002).

Conclusion

¹⁹F magnetic resonance angiography of intravenously administered perfluorocarbon emulsions is feasible for a selective and exclusive visualization of the vasculature in vivo.     

Fluorine-19 nuclear magnetic resonance studies of effects of ligands on trifluoroacetonylated supernatant aspartate transaminase.

The selective reaction of Cys-45 and -82, on the one hand, and Cys-390, on the other, with 3-bromo-1,1,1-trifluoropropanone allows for the probing of these regions of aspartate transaminase in the absence and in the presence of enzymatic ligands by 19F nuclear magnetic resonance (NMR). The 19F chemical shifts of the resonance lines differ for the three cysteines and so does their behavior with pH changes. The resonance signals with chemical shifts at 615 and 800 Hz upfield from trifluoroacetic acid correspond to modified cysteine-82 and -45 and have tentatively been assigned in this order. The 615-Hz resonance is affected by pH changes that fit best the influence of a single ionizing residue. On the 800-Hz line, the pH changes appear to be the influence of a minimum of two ionizing residues. The 19F resonance from modified Cys-390 is pH independent in the pH range 5-9 for the pyridoxal phosphate, pyridoxamine phosphate, and apoenzyme forms of the enzyme. Occupation of the active site by a quasi-enzyme-substrate complex, trifluoromethionine pyridoxyl phosphate, affects the 19F chemical shift of modified Cys-390, making it pH dependent with a pK value of 8.4. The 19F NMR properties of the pyridoxal form of Cys-390-modified enzyme can be used to monitor some ligand interactions with the active-center region. Addition of alpha-ketoglutarate or succinate to the ketone labeled enzyme causes a decrease in the resonance line width, and titrations show that this procedure is a good method with which to study the affinity of the enzyme for these ligands. The interpretation of the chemical shift and line-width characteristics of the 19F resonance arising from Cys-390 are most consistent with a model in which the region around this residue seems to be affected by conformational changes arising from substrate binding to the active-center subsites in productive (covalent) manner. Nonproductive complexes which possess fast ligand-protein exchange, such as those between alpha-ketoglutarate or succinate with the pyridoxal phosphate form of the enzyme, may result only in a greater degree of freedom for Cys-390.     

19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

The 19F NMR spectrum of Escherichia coli tRNA1Val in which [5-19F]uridine replaces 93% of all uridine and uridine-derived residues has been examined at 93.6 and 235 MHz. The resolution of 11 peaks and visibility of two additional shoulders at either frequency for the 14 FUra residues in the molecule attests to the excellence of 19F as a probe for the structure of tRNA1Val in solution. No significant gain in resolution was attained at the higher frequency. A comparison of the relative areas in the different regions of the 19F spectrum of mixed [FUra]tRNAs with that of [FUra]tRNA1Val suggests that the three single resonances at lowest field in the region 86.5 to 88.5 ppm upfield from trifluoroacetate correspond to the three invariant bases which form tertiary hydrogen bonds in all tRNAs, namely, 8 (U or s4U), 54 (T), and 55 (phi) in unsubstituted tRNAs.     

19F NMR of 5-fluorouracil-substituted transfer RNA transcribed in vitro: resonance assignment of fluorouracil-guanine base pairs.

5-Fluorouracil is readily incorporated into active tRNA(Val) transcribed in vitro from a recombinant phagemid containing a synthetic E. coli tRNA(Val) gene. This tRNA has the expected sequence and a secondary and tertiary structure resembling that of native 5-fluorouracil-substituted tRNA(Val), as judged by 19F NMR spectroscopy. To assign resonances in the 19F spectrum, mutant phagemids were constructed having base changes in the tRNA gene. Replacement of fluorouracil in the T-stem with cytosine, converting a FU-G to a C-G base pair, results in the loss of one downfield peak in the 19F NMR spectrum of the mutant tRNA(Val). The spectra of other mutant tRNAs having guanine for adenine substitutions that convert FU-A to FU-G base pairs all have one resonance shifted 4.5 to 5 ppm downfield. These results allow assignment of several 19F resonances and demonstrate that the chemical shift of the 19F signal from base-paired 5-fluorouracil differs considerably between Watson-Crick and wobble geometry.     

35Cl nuclear magnetic resonance study of zinc and phosphate binding of E. coli alkaline phosphatase

³⁵Cl^? quadrupole relaxation was measured in the presence of metal-free alkaline phosphatase and in the presence of Zn²⁺-alkaline phosphatase. The relaxation data show that for an enzyme containing the minimum amount of zinc needed for full activity—2 g atoms of zinc per mole of protein—there appears to be no binding of halide ions to the protein-bound zinc ions. In contrast, when there is a high metal-enzyme ratio, a large relaxation enhancement is observed, demonstrating coordination of halide ions to the metal ions.Addition of inorganic phosphate causes no change in the ³⁵Cl^? relaxation in the presence of metal-free enzyme. However, marked decreases in relaxation are observed upon addition of phosphate to the Zn²⁺-alkaline phosphatase. The relaxation measurements carried out in the presence of phosphate show that substrate binding does prove to be metal-ion dependent. Furthermore, experiments with inorganic phosphate suggest the tight binding of one phosphate to the alkaline phosphatase.     

Fluorine-19 as a covalent active site-directed magnetic resonance probe in aspartate transaminase.

Phosphypyridoxyl trifluoroethylamine has been synthesized as an active site-directed 19F NMR probe for aspartate transaminase. This coenzyme derivative adds stoichiometrically to the apotransaminase as observed by both fluorescence and circular dichroism measurements. The fluorinated phosphypyridoxamine derivative, when bound to the apotransaminase, will not dissociate upon extensive dialysis or passage through Sephadex G-25. The compound behaves as a pyridoxamine phosphate derivative and not as a coenzyme-substrate complex, since both competing anions and dicarboxylic acid inhibitors still bind to the phosphopyridoxyl trifluoroethylamine enzyme. The 19F NMR spectra of the enzyme-bound phosphopyridoxyl trifluoroethylamine were measured as a function of pH, ionic strength, and temperature. The 19F MNR of the enzyme-bound coenzyme derivative revealed no predetermined asymmetry in the subunits of aspartate transaminase insolution in terms of differences in chemical shift or resonance line shape between the two environments. A pH-dependent chemical shift change of the single 19F resonance was observed, which is consistent with the influence of a single ionization with an apparent pKa of 8.4 in 0.10 M KCl at 30 degrees. Increasing the ionic strength resulted in increasing values for the observed pKa, the highest recorded value was 9.1 in 3.0 M KCl. The temperature dependence of the pH titration of the chemical shift gives deltaH' of ionization of 10.5 kcal/mol. The evidence suggests a possible epsilon-amino group, electrostatically affected by positive charges, being responsible for the titration effect of the active site-bound fluorine derivative of pyridoxamine phosphate.     

Evaluation of base-pairing schemes for E. coli 5S RNA by 400 MHz proton nuclear magnetic resonance spectroscopy

The number of base pairs in the denatured “B” form of $\text{E. coli}$ 5S RNA has been determined directly from 400 MHz high resolution proton nuclear magnetic resonance spectroscopy. The experimental NMR spectrum from ?11.6 to ?14.5 ppm from a sodium 2,2-dimethyl-2-silapentane sulfonate reference can be simulated by a theoretical spectrum consisting of 33 Lorentzian lines of equal width (corresponding to 33 base pairs) at 26°C. This result is inconsistent with previously proposed secondary structures of 17 and 23 base pairs, but is readily adapted to the Luoma-Marshall cloverleaf secondary structure.     

Proton correlation nuclear magnetic resonance study of anaerobic metabolism of Escherichia coli.

Proton correlation nuclear magnetic resonance has been used to investigate anaerobic metabolism of glucose in Escherichia coli cells. The time course of the concentrations of six metabolites (ethanol, lactate, acetate, pyruvate, succinate, and formate) has been followed at the very early state of fermentation, and used to discuss dynamical aspects of the mixed-acid fermentation of glucose by E. coli.     

Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard.     

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca.

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.