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SynopsisThe high-resolution (300 MHz) proton nmr spectrum of E. coli tRNAfMet has been examined in 0.17M NaCl, with and without hIg2+, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNAfMet, some low field (1 1-15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure Alr-S4U8 base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned t o secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mgz+ the tertiary structure base pair is present only to -45"C, but in the presence of Mg2+ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A14-S4U8 base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mgz+.

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High-resolution 300-MHz proton nuclear magnetic resonance spectra of the hydrogen-bonded protons in three different purified tRNA molecules are presented. The resonances in the region between -11 and -15 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) are assigned to the ring NH protons of specific base pairs by two approaches. First, intrinsic positions of -14.8 ppm and -13.7 ppm are taken for the AU and GC ring NH protons, respectively, and the spectra are calculated by including ring current shifts from the nearest neighbors.The spectra calculated in this way on the basis of the cloverleaf are in good agreement with the observed. Second, fragments of yeast tRNAPhe were obtained, which helped in assignments of the spectrum of intact molecules. The close agreement strongly supports the cloverleaf model.Tertiary structural features were determined in a few cases where the ring currents at the terminal base pairs of helical regions depended upon stacking of the helices.In this way, we were able to show that in Escherichia colt tRNAGlu the CCA stem forms a continuous helix with the TVt-C stem, which is in accord with the preliminary x-ray structure of yeast tRNAPhe, suggesting that this stacking is observed in solution and may be a general property of different tRNA molecules. Similar reasoning suggests that in E. coli tRNAfMet G-27 is stacked upon the dihydrouridine helix.In order to understand in detail how tRNA functions in protein synthesis, the structure of tRNA in solution must be determined. The present note summarizes results of our recent high-resolution proton nuclear magnetic resonance (NMR) investigations (1-4) of tRNA in which we determined that the cloverleaf model is an accurate description of the hydrogen-bonded secondary structure of several purified tRNAs, including yeast tRNAPhe, Escherichia coli tRNAGlU, and E. coli tRNAfMet. In addition, we obtained information about certain important tertiary structural features of each molecule. In this paper we emphasize these structural aspects to show how the NMR measurements in solution complement and supplement the crystal structure presently emerging from the x-ray studies (5).The groundwork for the present study was developed in previous studies of the NMR spectra of tRNA molecules in solution where we showed that resonances observed in the region between -11 and -15 ppm from 2,2-dimethyl-2-Abbreviations: NMR, nuclear magnetic resonance; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate.

The effect of aminoacylation on the conformation of yeast tRNAPhe was investigated by highresolution (300 MHz) proton nuclear magnetic resonance (NMR) spectroscopy. Resonances in the low-field (-11 to -15 ppm) region of the spectra are due to ring NH protons of Watson-Crick base pairs, and to a very high degree of approximation (within 0.05 ppm) the low-field spectra of tRNAPhe and phenylalanyl-tRNAPhe are identical. From this observation and analysis of the low-field NMR spectra we conclude that the secondary structures of the two tRNAs are identical with respect to base-pairing schemes and interbase distances in the helical region (0.1-0.2 A). Several tertiary structural features, including conformation of the dihydro-U loop, conformation of the minor loop, relative orientations of the acceptor and the TV&C stems, dihydro-U and anticodon stems, and probably conformation of the anticodon loop are shown to be the same in tRNAPhe and phenylalanyl-tRNAPhe. Our results leave little remaining opportunity for changes in tertiary structure that would not have been observed by the NMR method.In order to account for the role that tRNA plays in protein synthesis, it has been suggested that aminoacylation (or deacylation) induces a change in the secondary and/or tertiary structure of the tRNA (1-3). During the past 7 years, there have been numerous attempts to investigate this problem, but in each case, the methods used were limited in the information they provided and the results were often conflicting. Sarin and Zamecnik (1), for example, reported significant differences between the optical rotatory dispersion of unfractionated aminoacyl-tRNA and tRNA, but Adler and Fasman (4) used purified tRNAs and found no difference in the circular dichroism of aminoacyl-tRNA and tRNA. Gantt, Englander, and Simpson (5) found differences between the hydrogen-exchange properties of unfractionated Escherichia coli tRNA before and after aminoacylation, but the differences in the number of exchangeable protons detected by the tritium exchange technique was small (order of 4%). In a subsequent study of the hydrogen-exchange properties of E. coli tRNAfMet, Englander, Kallenbach, and Englander (6) were unable to detect any difference between aminoacylated and deacylated tRNA.Hanggi and Zachau (7) were unable to detect any difference in gel filtration or in partial nuclease digestion of tRNA and aminoacylated tRNA, and concluded that there were no gross conformational changes in tRNA induced by aminoacylation. Cohn, Danchin, found that binding of Mn2+ to tRNA was increased by aminoacylation of tRNA, but did not speculate on possible conformational changes that might be involved. Danchin and Grunberg-Manago (9) found that the binding of oligo (C) to unfractionated E. coli tRNA, purified yeast tRNAPhO, and E. coli tRNAval is stronger when the tRNAs are aminoacylated, and concluded from this that a region of the tRNA containing at least two guanine residues was exposed in the aminoacylated tRNAs but not in the uncharged tRNA. They furth...