Hydrogen exchange study of some polynucleotides and transfer RNA

Abstract

The apparent disagreement between published transfer RNA hydrogen exchange results and the tRNA cloverleaf model, prompted a re-investigation of the relationship between hydrogen exchange data and nucleic acid structure. Hydrogen-tritium exchange experiments were carried out with samples of pure and mixed tRNA and with the synthetic polynucleotide bihelices: poly(rA) · poly(rU), poly(rI) · poly(rC), poly(rG) · poly(rC) and poly(dG) · poly (dC). Studies With the synthetic polynucleotides show that, to interpret nucleic acid hydrogen exchange data in terms of quantity of base-paired structure, one must count 5 H for each G · C pair and 2 or 3 for A · U. Both poly(rG) · poly(rC) and poly(dG) · poly(dC) clearly show 5 slowly exchanging H per base pair. For A · U and I · C only 2 were detected, though other workers have found 3 for some A-T systems. These are all base-pair-bound H. The ribose OH is too fast to measure. The reasons for the surprisingly slow exchange of the exposed NH 2 protons are unknown. The hydrogen exchange-rate behavior found for the polynucleotides suggests that some local structural distortion is necessary for any of the exchangeable H to react, including the exposed NH 2 protons, and that the distortion important for hydrogen exchange is different from that occurring in thermal denaturation. All the tRNA samples show very similar hydrogen exchange profiles. The pure samples (formylmethionine and tyrosine tRNA from Escherichia coli) have ~120 slowly exchanging protons, far more than the ~55 Watson-Crick hydrogen bonds in the simple cloverleaf models. With the above numeration, however, the cloverleaf models for the two pure tRNA samples account for all but approx-imately 20 of their slowly exchanging H. The excess of 20 H is very close to the number required by models having extra tertiary structure. The tRNA's were found to exchange more slowly even than poly(rG) · poly(rC) and to have a unique salt and pH dependence. These anomalies could also be explained by the presence of some tertiary folding. Unacylated and 70% aminoacylated E. coli tRNA f Met were found to have identical hydrogen exchange behavior, suggesting absence of structure change upon aminoacylation. A method was developed for isolating authentic poly(rG) from normally heterodisperse mixtures by Sephadex gel filtration in 90% dimethyl sulfoxide. Formation of the 1:1 poly(rG) · poly(rC) complex was achieved by mixing-experiments in concentrated urea solutions at high temperature.