Quantitative determination of the number of secondary and tertiary structure base pairs in transfer RNA in solution

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Abstract
Resonances in the low-field (11-15 ppm) NMR spectrum of tRNA molecules arise from secondary and tertiary structure base pairs (1 resonance for each base pair) as well as tertiary structure H-bonds. An accurate method for integrating the low-field spectra was developed and applied to 7 different tRNA [yeast and Escherichia coli]. In the presence of high levels of Mg (10 mM free Mg) the number of resonances (base pairs) per molecule is typically 3-4 more than the number predicted by the cloverleaf model. These results confirm the recent proposal that, under proper conditions, most tRNA exhibit 3-4 tertiary structure interactions in solution, which are also observed in X-ray diffraction studies of yeast tRNAPhe. In addition to common resonances in the 11-15 ppm region, there are common resonances at 10.5 and 9.5 ppm. A critique of methods used to integrate the low-field spectra is given and possible sources of error are indicated. The discrepancy between the present results and previous studies, which indicated that the number of base pairs per molecule was close to the number predicted by the cloverleaf model, can be attributed partly to differences in Mg concentration and partly to inaccuracies inherent in the integration methods used.