Tertiary hydrogen bonds in the solution structure of transfer RNA

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 tRNA1-Va1 indicate that there are 26 plus or minus 3 protons, while only 20 are expected from secondary structure Watson-Crick hydrogen bonds inthe 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 tRNA1Va1, E. coli tRNA-Arg and E. coli tRNA-Phe have one "GU pair" in their cloverleaf structure, while the fourth, yeast tRNA-Asp,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 tRNA-Phe. This assignment is confirmed by the disappearance of this resonance after treatment with cyanogen bromide.