Fluorine-19 nuclear magnetic resonance study of codon-anticodon interaction in 5-fluorouracil-substituted E. coli transfer RNAs.

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.     

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.     

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.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

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 tRNAVal1, 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) (delta FUra = -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.     

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.     

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.     

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)     

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.     

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).     

Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.     

Characterization of the fluorodihydrouracil substituent in 5-fluorouracil-containing Escherichia coli transfer RNA

The fluorodihydrouridine derivative previously detected in one of two isoaccepting forms of FUra-substituted Escherichia coli tRNA^Met_f 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, tRNA^Met_m and tRNA^Val_l, 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) (δ_FUra = ? 31.4 ppm; J_HF = 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 (J_HF = 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.     

A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome

Ribosome complexes containing deacyl-tRNA1(Val) or biotinylvalyl-tRNA1(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the tRNA1(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450-5454]. However, in complexes containing deacyl-tRNA1(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA1(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.     

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.     

Recognition of Escherichia coli valine transfer RNA by its cognate synthetase: a fluorine-19 NMR study.

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.     

Effect of zinc ions on tRNA structure: imino proton NMR spectroscopy

The structure of tRNA in solution was explored by NMR spectroscopy to evaluate the effect of divalent cations, especially zinc, which has a profound effect on the chromatographic behaviour of tRNAs in certain systems. The divalent ions Mg2+ and Zn2+ have specific effects on the imino proton region of the 1H NMR spectrum of valine transfer RNA (tRNA(Val] of Escherichia coli and of phenylalanine transfer RNA (tRNA(Phe] of yeast. The dependence of the imino proton spectra of the two tRNAs was examined as a function of Zn2+ concentration. In both tRNAs the tertiary base pair (G-15).(C-48) was markedly affected by Zn2+ (shifted downfield possibly by as much as 0.4 ppm); this is the terminal base pair in the augmented dihydrouridine helix (D-helix). Base pair (U-8).(A-14) in yeast tRNA(Phe) or (s4U-8).(A-14) in tRNA1(Val), which are stacked on (G-15).(C-48), was not affected by Zn2+, except when 1-2 Mg2+ ions per tRNA were also present. Another imino proton that may be affected by Zn2+ in both tRNAs is that of the tertiary base pair (G-19).(C-46). The assignment of this resonance in yeast tRNA(Phe) is tentative since it is located in the region of highly overlapping resonances between 12.6 and 12.3 ppm. This base pair helps to anchor the D-loop to the T psi C loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complementary addressed modification of yeast tRNA Val 1 with alkylating derivative of d(pC-G)-A. The positions of the alkylated nucleotides and the course of the alkylation in the complex.

Yeast tRNA Val 1 alkylation with 2', 3'-O-4-(N-2-chloroethyl-N-methylamino) benzylidene d(pC-G)-A proceeds at 20 degrees - 30 degrees C in the complementary complexes which are formed by d(pC-G)-A greater than RC1 binding to 3 sequences of tRNA Val 1 : psi-C-G58 in the T loop, C-G40 at the 3'-side of the anticodon loop and C-G18 in the D loop. The reaction in the complexes results in A53, I35, and psi 13 alkylation to form beta-/N-methyl-N-(formylphenyl 17 amino/ethyl-tRNA Val 1 with the relative rate constants of the alkylation that are 3 or 2 orders of magnitude higher than that for the alkylation without a complex formation. It is the third nucleotide from the 5'-terminus of the binding site of the modifying agent that is subjected to alkylation in the t RNA Val 1. The course of the alkylation does not depend on the possible base pairing of the 3'-terminal nucleotide of the reagent. The extent of the reagent binding and the relative rate constants of the alkalytion in the complexes indicate the following order of the complex stability: (psi-C-G58) greater than (CO-G40) approximately (C-G18) at 20 degrees and (psi-C-G58) greater than (C-G40) greater than (C-G18) at 30 degrees.     

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)