Yeng P. Wong

Ring-current shifts in the 300 MHz nuclear magnetic resonance spectra of six purified transfer RNA molecules

Abstract We present the 300 MHz high-resolution proton nuclear magnetic resonance spectra of the ring NH hydrogen-bonded protons of six purified tRNAs. Good agreement was obtained between the observed spectra and those computed on the assumption of the suitable cloverleaf models. In the computation it is assumed that the hydrogen-bonded ring NH in each type of base pair has an intrinsic position with respect to 2,2-dimethyl-2-silapentane-5-sulfonate, i.e. in A·U it is at −14·8 parts per million, in G·C at −13·7 parts per million and in A·Ψat −13·5 parts per million. The shifts of these resonances from these positions are calculated by including ring current fields from the nearest neighbors. The agreement is very good, adding support to our earlier findings that there is no evidence for additional Watson-Crick base pairs detected beyond those in the cloverleaf. In general, resolved resonances are fitted by the computed spectra to within ±0·2 part per million showing that there is no need for any additional physical mechanism to explain the nuclear magnetic resonance positions. Hence, the nuclear magnetic resonance spectra can be interpreted in terms of the structure of their neighbors and in a few important cases this has been particularly valuable in understanding the structure beyond the end of a helical region. In the tRNA Glu E.coli′ for example, the positions of the resonances in A·U no. 7 and A·U no. 49 at the interior ends of the acceptor and -T-Ψ-C- stems, respectively, strongly suggest that these two stems are in a continuous helix. Other structural effects at the ends of the helical regions are also suggested by the nuclear magnetic resonance spectra.

Investigation of exchangeable protons and the extent of base pairings in yeast phenylalanine transfer RNA by high resolution nuclear magnetic resonance

Abstract High resolution nuclear magnetic resonance spectra were obtained for yeast transfer RNA Phe and unfractionated yeast tRNA in aqueous solutions. Resonances due to the free amino protons, hydrogen-bonded amino protons and hydrogen-bonded ring N—H protons were observed in three non-overlapping spectral regions. The assignment of the exchangeable proton resonances in yeast tRNA Phe is based on their pH and temperature dependence, spectral position, and their disappearance in deuterium oxide solvent. The number of each kind of exchangeable proton was determined and compared with the number expected on the basis of the cloverleaf model. The total number of base pairs observed by nuclear magnetic resonance (20±2) agrees well with the number computed from the cloverleaf model (20±1) depending upon whether or not one 2MeG.C and one G.U base pair are included. Furthermore, the separate numbers of A.U and G.C base pairs, 7 to 8 and 11 to 12, respectively, determined by nuclear magnetic resonance, are in good agreement with the 8 and 12 respective pairs expected from the cloverleaf model. The nuclear magnetic resonance spectra provided little evidence for additional strong Watson-Crick type base pairs resulting from tertiary structure such as are proposed in several models of tRNA. Resonances observed in the −9 to −11 parts per million region appear to be related to tertiary structure, but their assignment remains open at this tune. The results obtained with unfractionated yeast tRNA parallel those obtained with yeast tRNA Phe and therefore indicate that the interpretation of these latter data may be applicable to other tRNA species. The high resolution nuclear magnetic resonance data on the behavior of the exchangeable protons in tRNA are compared with the results of recent tritium exchange experiments. The anomalous behavior of hydrogen-bonded amino protons in both the nuclear magnetic resonance and tritium exchange experiments is discussed.

Investigation of the secondary structure of Escherichia coli 5 S RNA by high-resolution nuclear magnetic resonance☆

The base-pairing structure of Escherichia coli 5 S RNA has been studied by high-resolution proton nuclear magnetic resonance spectroscopy. In the first analysis, these experiments provide a direct measure of the A · U and G · C base-pair content. Measurements over a range of temperatures indicate that the number of base pairs decreases from 28 ± 2 at 30 °C to 16 to 18 in the 52 °C to 62 °C range. On heating to 72 °C, this number is further reduced to about eight. An analysis of the nuclear magnetic resonance spectra in terms of nearest-neighbor ring current field affects on the positions of the low-field proton resonances provides a highly detailed picture of the 5 S RNA in its different stages of melting. The nuclear magnetic resonance spectra of all possible helices containing three or more base-pairs were computed and used in conjunction with the observed spectral data, general thermodynamic considerations and observations on the spectra of other polynucleotides to identify the helical sections present in 5 S RNA at 50 to 60 °C. This analysis leads to a model that includes three helices containing ten, seven and four base-pairs, respectively. If the helices that are present at the high temperatures are assumed to be present at lower temperatures, then the base-pairing matrix shows that many other helices are automatically excluded from consideration. This information, along with the nuclear magnetic resonance difference spectrum (30 to 50 °C), is sufficient to identify the two helices that are added when the temperature is lowered to 30 °C. In this way, a model for the complete secondary structure of 5 S RNA at room temperature can be deduced, which is consistent with other chemicals and phyical data. None of the previously proposed models are consistent with the nuclear magnetic resonance data.

The extent of base pairing in 5 s RNA Yeast 5 s RNA

Abstract The total number of secondary structure base pairs in yeast 5 s RNA has been determined by measuring the integrated intensities of the proton nuclear magnetic resonance in the −11 to −15 part per million region. In the absence of Mg2+ 21 ± 2 base pairs were observed at 35 °C. After incubating in the presence of Mg2+ the total base-pair content in yeast 5 s RNA increased to 28 ±3, most of which (85%) are G.C pairs. The small fraction of base pairing indicated by the nuclear magnetic resonance experiments is supported by comparative enzymic digestion studies which show that yeast 5 s RNA is considerably more susceptible to degradation than is transfer RNA.

On the tautomeric states of guanine and cytosine

Abstract Recently Chan and coworkers reported that the H8 proton of guanine and H5 proton of cytosine have unusually broad resonances in the PMR spectrum under certain conditions of temperatures and pD, and interpreted their results as indicating that the minor tautomers of guanine and cytosine were present to the extent of 15 ± 3%. We find, however, that the previously reported phenomena are not reproducible with purified samples of 2′ GMP and 5′ CMP, but with addition of paramagnetic impurities the pD and temperature dependence of GH8 and CH5 proton linewidth broadening previously reported can be reproduced. This study, therefore, raises serious questions about the existence of 15% minor tautomers in cytosine and guanine at room temperature in neutral aqueous solution.