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.