Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.
The effects of different cations on the hydrodynamic radius (RH) of a 48-bp synthetic DNA are measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium. Relative statistical errors in RH are only approximately 1%. With increasing cation concentration, Na+ causes a small decrease in RH, Cs+ causes a somewhat larger decrease by up to 0.5 A at 100 mM, and (CH3CH2)4N+ causes an increase in RH by approximately 0.5 A at 100 mM. The qualitatively different effects of these monovalent cations indicates that the changes in RH with cation concentration do not arise primarily from electrolyte friction. Divalent cations cause much larger increases in RH with increasing cation concentration. Mg2+ causes an increase in RH by up to 1.0 A at 24.4 mM, and Mn2+ causes an increase in RH by up to 1.6 A at 24.4 mM. These effects are independent of DNA concentration. There is some positive correlation between the order of effects of the different cations on RH and the order of their effects on interhelical hydration forces. It is suggested that these different ions affect RH either by altering the hydration layer or possibly by some effect on DNA structure, such as stabilizing bends.
The high resolution nuclear magnetic resonance (NMR) spectra of hydrogen-bonded protons in four tRNAs have been studied at 270 MHz. The relative intensity of the resonances between -11 ppm and -15 ppm of Escherichia coli tRNAlV1l indicate that there are 26 4 3 protons, while only 20 are expected from secondary structure Watson-Crick hydrogen bonds in the cloverleaf structure. Several possible candidates for these extra resonances are suggested by tertiary interactions observed in recent crystallographic studies.Of the four tRNAs studied, three, e.g., E. coli tRNAlva1, E. coli tRNA-'s and E. coli tRNAPhe have one "GU pair' in their cloverleaf structure, while the fourth, yeast tRNAIP, has three "GU pairs" and one "G' pair". Correlating these with the NMR spectra in the -10 ppm to -11 ppm region allows us to conclude that the "GU pairs" are not hydrogen-bonded by tautomerization to the lactim form.At the very low field region, near -14.9 ppm, the three E. coli tRNAs show a single resonance which is attributed to the 4-thiouracil 8 to adenine 14 hydrogen bond of the tertiary structure, by analogy with the recent crystal structure of yeast tRNAPhe. This assignment is confirmed by the disappearance of this resonance after treatment with cyanogen bromide.Previous studies indicate that the low field (-11 to -15 ppm) nuclear magnetic resonance (NMR) spectra of nucleic acids contain contributions from one proton in the ring NH hydrogen bond of each base pair (1-3) of the secondary structure.Recent studies on Eicherichia coli tRNAGlU assigned one addcitional resonance to a tertiary structure hydrogen bond (4); however, the numerous hydrogen bonds from tertiary interaction seen in the recent x-ray crystal structures (5, 6) have not previously been apparent in the NMR spectra of the tRNAs. It is obvious that NMR spectra of tRNAs with greater resolution would allow a more reliable assignment of the resonances, including resonances in the low field region from other kinds of exchangeable protons, as well as possible additional resonances from the tertiary structure. Assignment of the resonances in the yeast tRNAPhe spectrum (3), together with the spectra of other tRNA species, led to the generation of a set of ring current shift rules which approximately predict the observed resonance positions (7). These assignments have been strengthened by recent NMR measurements at various temperatures where sequential melting of several different tRNAs was observed (4, 8). The resulting first order understanding of the low field resonance positions allowed us to identify tRNA species of known sequence which were predicted to give inherently better resolved spectra; such well-resolved spectra would hopefully reveal additional tertiary resonances if they were present. In the present paper Abbreviations: NMR, nuclear magnetic resonance. 2049we take advantage of the better spectral resolution obtained by the use of highly purified tRNA samples, some which have not been studied before, and improved magnetic field homogeneity at 270 M...
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