Determination of Secondary and Tertiary Structural Features of Transfer RNA Molecules in Solution by Nuclear Magnetic Resonance

Abstract

High-resolution 300-MHz proton nuclear magnetic resonance spectra of the hydrogen-bounded 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 tRNA(Phe) 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 coli tRNA(Glu) the CCA stem forms a continuous helix with the TPsiC stem, which is in accord with the preliminary x-ray structure of yeast tRNA(Phe), 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 tRNA(fMet) G-27 is stacked upon the dihydrouridine helix.