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1.
Jack Horowitz Matthew L. Cotten Charles C. Hardin Paul Gollnick 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1983,741(1):70-76
The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNAMetf has been further characterized. This substituent is responsible for the 19F resonance observed 15 ppm upfield from free FUra (= 0 ppm) in the high resolution 19F-NMR spectra of FUra-substituted tRNA purified by chromatography on DEAE-cellulose, at pH 8.9, to remove normal tRNA. Similar highfield 19F signals have now been observed in the spectra of two other purified fluorinated E. coli tRNAs, tRNAMetm and tRNAVall, as well as in unfractionated tRNA, indicating the widespread occurrence of the constituent. Comparison with 19F spectrum of the model compound 5′-deoxy-5-fluoro-5,6-dihydrouridine (dH56FUrd) (δFUra = ? 31.4 ppm; JHF = 48 Hz) indicates that the substituent does not contain an intact fluorodihydrouridine ring. dH56FUrd is considerably more alkali labile than 5,6-dihydrouridine (H56Urd). At pH 8.9, where H56Urd is stable, dH56FUrd is degraded to a derivative, presumably a fluoroureidopropionic acid, with a 19F resonance at ? 15.7 ppm that nearly coincides with the upfield peak in the spectrum of pH 8.9-treated tRNA. The 19F-NMR spectrum of fluorinated tRNA, not exposed to pH 8.9, exhibits two peaks 31 and 32 ppm upfield of FUra, in place of the 19F signal at ? 15 ppm. Hydrolysis of this tRNA with RNAase T2 produces a sharp doublet 33 ppm upfield (JHF = 45 Hz). Similarities of the 19F chemical shift and coupling constant to those of dH56FUrd, allows assignment of the peak at ? 33 ppm to an intact fluorodihydrouridine residue in the tRNA. Our results demonstrate that FUra residues incorporated into E. coli tRNA at sites normally occupied by dihydrouridine can be recognized by tRNA-modifying enzymes and reduced to fluorodihydrouridine. This substituent is labile at moderately alkaline pH values and undergoes ring-opening during purification of the tRNA. 相似文献
2.
Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids 总被引:3,自引:0,他引:3
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. 相似文献
3.
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. 相似文献
4.
Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs. 总被引:2,自引:2,他引:0
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Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAVal1 (anticodon FAC) by 19F NMR spectroscopy. Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9 ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNATyr2 (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAVal1. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMetm. However, ApUpG has no effect on the 19F spectrum of 19F-labeled E. coli tRNAMetf, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of CpGpApA to the complementary FpFpCpG (replaces Tp psi pCpG) in the T-loop of 5-fluorouracil-substituted tRNAVal1, in the presence or absence of codon, suggesting that the tertiary interactions between the T- and D-loops are not disrupted by codon-anticodon interactions. 相似文献
5.
Fluorine-19 nuclear magnetic resonance studies of the structure of 5-fluorouracil-substituted Escherichia coli transfer RNA 总被引:1,自引:0,他引:1
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) 相似文献
6.
19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA 总被引:1,自引:0,他引:1
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. 相似文献
7.
In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells 总被引:4,自引:0,他引:4
19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C. 相似文献
8.
19F NMR of 5-fluorouracil-substituted transfer RNA transcribed in vitro: resonance assignment of fluorouracil-guanine base pairs. 总被引:4,自引:2,他引:2
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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. 相似文献
9.
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). 相似文献
10.
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. 相似文献
11.
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. 相似文献
12.
Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study. 总被引:1,自引:0,他引:1
Interactions of 5-fluorouracil-substituted Escherichia coli tRNAVal with its cognate synthetase have been investigated by fluorine-19 nuclear magnetic resonance. Valyl-tRNA synthetase (VRS) (EC 6.1.1.9), purified to homogeneity from an overproducing strain of E. coli, differs somewhat from VRS previously isolated from E. coli K12. Its amino acid composition and N-terminal sequence agree well with results derived from the sequence of the VRS gene [Heck, J.D., & Hatfield, G.W. (1988) J. Biol. Chem. 263, 868-877]. Apparent KM and Vmax values of the purified VRS are the same for both normal and 5-fluorouracil (FUra)-substituted tRNAVal. Binding of VRS to (FUra)tRNAVal induces structural perturbations that are reflected in selective changes in the 19F NMR spectrum of the tRNA. Addition of increasing amounts of VRS results in a gradual loss of intensity at resonances corresponding to FU34, FU7, and FU67, with FU34, at the wobble position of the anticodon, being affected most. At higher VRS/tRNA ratios, a broadening and shifting of FU12 and of FU4 and/or FU8 occur. These results indicate that VRS interacts with tRNAVal along the entire inside of the L-shape molecule, from the acceptor stem to the anticodon. Valyl-tRNA synthetase also causes a splitting of resonances FU55 and FU64 in the T-loop and stem of tRNAVal, suggesting conformational changes in this part of the molecule. No 19F NMR evidence was found for formation of the Michael adduct between VRS and FU8 of 5-fluorouracil-substituted tRNAVal that has been proposed as a common intermediate in the aminoacylation reaction. 相似文献
13.
Halogenated nucleic acids: effects of 5-fluorouracil on the conformation and properties of a polyribonucleotide and its constituents 总被引:2,自引:0,他引:2
The conformational properties of 5-fluorouracil derivatives are compared to uracil derivatives. FUrd, 5′-FUMP, and poly(FU) are studied as a function of pH and temperature by 19F- and 1H-nmr spectroscopy, and the corresponding uracil derivatives by 1H-nmr spectroscopy. FUrd exhibits no significant conformational changes with solution pH (5–10). In contrast, at low pH (6–7) 5′-FUMP and 5′-UMP show similar conformational features, while at high pH (9) 5′-FUMP shows significant conformational alterations. Also, poly(U) and poly(FU) are conformationally similar at low pH, but increasing pH induces changes in poly(FU). These changes are observed in the backbone [γ(C4′-C5′)], furanose, and furanose-base conformations. The apparent pKa of N3-H ionization of the FUra base is determined by 1H- and 19F-nmr to range from 7.5 to 8.2 [FUrd < 5′-FUMP < 5′-FUDP < poly(FU)]. These observations are interpreted as a result of electrostatic interactions generated between the ionized phosphate group and the negatively charged base moiety as the pH is raised. The interaction properties of poly(FU) with ApA are studied by 1H- and 19F-nmr spectroscopy, and these properties compared to those published for poly(U). Poly(FU) forms a complex with ApA inducing upfield 1H-shifts in both components, and downfield 19F- shifts in poly(FU). The base stoichiometry of the complex for poly(U)·ApA is 2U:1A at various U/A ratios. In contrast, the base stoichiometry of the poly(FU)·ApA complex appears to be dependent on the FU/A ratio. At high FU/A ratio, the complex is 2FU:1A, and as the FU/A ratio approaches unity the complex becomes 1FU:1A. 相似文献
14.
15.
16.
On the mechanism of the acid-catalyzed hydrolysis of uridine to uracil. Evidence for 6-hydroxy-5,6-dihydrouridine intermediates 总被引:2,自引:0,他引:2
In acidic media, the 5,6-double bond of uridine is rapidly hydrated to give a small amount of 6-hydroxy-5,6-dihydrouridine (Urd-H2O), the mechanism of which is known from studies of the acid-catalyzed dehydration of Urd-H2O (Prior, J. J., Maley, J., and Santi, D. V. (1984) J. Biol. Chem. 258, 2422-2428). In addition to dehydration, Urd-H2O also undergoes direct hydrolysis of the N-glycosidic bond in acidic solution. The kinetics of the above reaction demonstrates that Urd-H2O, or an intermediate in the pathway leading from Urd to Urd-H2O, is kinetically competent to account for the hydrolysis of the N-glycosidic bond of Urd. The hydrolysis of (1'-2H)Urd proceeds with an alpha-secondary deuterium isotope effect of kH/kD of 1.11 at 25 degrees C. This isotope effect is sufficiently large to implicate carbonium ion character at the 1'-carbon during hydrolysis but, since it is not the maximal value expected, suggests that N-glycoside cleavage is rate-determining with a transition state intermediate between reactant and products. Importantly, the hydrolysis of [6-3H]Urd proceeds with a substantial inverse secondary isotope effect of kT/kH = 1.15 at 25 degrees C which indicates some degree of sp2 to sp3 rehybridization of C-6 of the pyrimidine moiety during hydrolysis. From the data available, it appears that an important pathway in the hydrolysis of the N-glycoside bond of Urd involves either spontaneous cleavage of Urd which is protonated at the 5-carbon or a protonated species of Urd-H2O. The studies described here, together with the known susceptibility of the 6-position of pyrimidine heterocycles toward nucleophiles, permits the proposal of chemically reasonable mechanisms for enzyme-catalyzed cleavage of N-glycosidic bonds of pyrimidines. 相似文献
17.
Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli 总被引:4,自引:0,他引:4
During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene. 相似文献
18.
Michael J. Beckage Michael Blumenstein Roy L. Kisliuk 《Molecular and cellular biochemistry》1980,32(1):45-48
Summary In the presence of thymidylate synthase, the 19F signal of 5-fluoro-2-deoxyuridylate is shifted upfield 0.6 ppm or 4.5 ppm depending on the enzyme preparation used. The bands at these positions represent different species of binary complex. When either binary complex is reacted with methylenetetrahydrofolate a ternary complex is formed with a 19F signal shifted 12.5 ppm upfield and broadened to 120 Hz.Substitution of the hydrogen atoms of the methylene group of methylenetetrahydrofolate with deuterium atoms results in line-narrowing of the spectrum of the ternary complex from 120 to 80 Hz indicating the close proximity of the methylene group to the fluorine atom in the ternary complex. A model compound, 5-fluoro-6-hydroxy-5-methyl-5, 6-dihydrouracil, gives a chemical shift in the same direction and of similar magnitude to that seen with the ternary complex. 相似文献
19.
Aspartate transcarbamoylase labeled with 3-fluorotyrosine was purified from an Escherichia coli strain which was auxotrophic for tyrosine and overproduced aspartate transcarbamoylase upon uracil starvation. The labeled enzyme in which about 85% of the tyrosines were replaced by fluorotyrosine exhibited high enzyme activity that varied in a sigmoidal manner with respect to the aspartate concentration. Also, the labeled enzyme was inhibited by CTP, activated by ATP, and exhibited a 2.6% decrease in sedimentation coefficient upon the addition of the active-site ligand, N-(phosphonacetyl)-L-aspartate. Thus, despite extensive replacement of tyrosines by fluorotyrosine, the modified enzyme was similar to native aspartate transcarbamoylase. The 19F nuclear magnetic resonance spectrum of isolated regulatory subunits labeled with fluorotyrosine consisted of a single peak. Addition of the activator, ATP, or the inhibitor, CTP, caused a loss of intensity at about 61.3 ppm upfield from a trifluoroacetic acid reference and an increase at about 61.5 ppm, but CTP also caused an increase at about 61.0 ppm. Five overlapping resonances were observed in the 19F NMR spectrum of unliganded catalytic subunits containing fluorotyrosine. Although the binding of the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, or the combination of carbamoylphosphate and succinate caused similar disappearances of resonances, the addition of N-(phosphonacetyl)-L-aspartate caused the appearance of resonances not observed with carbamoylphosphate plus succinate. Carbamoylphosphate alone perturbed three or four resonances and the subsequent addition of succinate affected at least two. 相似文献
20.
Catalytic mechanism and inhibition of tRNA (uracil-5-)methyltransferase: evidence for covalent catalysis 总被引:9,自引:0,他引:9
tRNA (Ura-5-)methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine (AdoMet) to the 5-carbon of a specific Urd residue in tRNA. This results in stoichiometric release of tritium from [5-3H]Urd-labeled substrate tRNA isolated from methyltransferase-deficient Escherichia coli. The enzyme also catalyzes an AdoMet-independent exchange reaction between [5-3H]-Urd-labeled substrate tRNA and protons of water at a rate that is about 1% that of the normal methylation reaction, but with identical stoichiometry. S-Adenosylhomocysteine inhibits the rate of the exchange reaction by 2-3-fold, whereas an analogue having the sulfur of AdoMet replaced by nitrogen accelerates the exchange reaction 9-fold. In the presence (but not absence) of AdoMet, 5-fluorouracil-substituted tRNA (FUra-tRNA) leads to the first-order inactivation of the enzyme. This is accompanied by the formation of a stable covalent complex containing the enzyme, FUra-tRNA, and the methyl group of AdoMet. A mechanism for catalysis is proposed that explains both the 5-H exchange reaction and the inhibition by FUra-tRNA: the enzyme forms a covalent Michael adduct with substrate or inhibitor tRNA by attack of a nucleophilic group of the enzyme at carbon 6 of the pyrimidine residue to be modified. As a result, an anion equivalent is generated at carbon 5 that is sufficiently reactive to be methylated by AdoMet. Preliminary experiments and precedents suggest that the nucleophilic catalyst of the enzyme is a thiol group of cysteine. The potent irreversible inhibition by FUra-tRNA suggests that a mechanism for the "RNA" effects of FUra may also involve irreversible inhibition of RNA-modifying enzymes. 相似文献