PROTON MAGNETIC RELAXATION STUDIES OF MANGANOUS COMPLEXES OF TRANSFER RNA AND RELATED COMPOUNDS

The interaction of transfer RNA and related compounds with metal ions has been investigated by measuring the enhancement of the effect of the paramagnetic manganous ions, when bound, upon the longitudinal relaxation rate of the solvent water protons; the enhancement parameter reflects dynamic properties of the macromolecular structure at the binding site of the manganous ions. It has been found that for unfractionated tRNA of Escherichia coli and purified tRNAPhe of E. coli there is one class of interacting strong binding sites (KA = 2 to 4 × 104m) representing 0.08 site per phosphate, n1, characterized by an enhancement factor, ε{lunate}b1, of 19.2 ± 1.0. Any modification of tRNA altered the value of the enhancement parameter. Upon aminoacylation, n1 was unchanged but ε{lunate}b1 decreased to 13.0 and 12.5, respectively, for unfractionated tRNA and tRNAPhe reflecting a change in structure at the six to seven strong binding sites per molecule. Fragments of tRNA, chain length approximately 40 nucleotides, obtained after hydrolysis by sheep kidney nuclease exhibited greatly altered binding properties since the structural parameter, ε{lunate}b1, was lowered to 6.0 and the number of sites, n1, increased to 0.13. Similarly, for denatured leucine tRNAIII of yeast, ε{lunate}b1 was lowered to 8.0 and for denatured unfractionated tRNA of E. coli, the average value of ε{lunate}b1 was 13.0. In addition to the small number of co-operative strong binding sites for manganese in tRNA, another class of weaker sites were observed equivalent to approximately 0.22 site per phosphate residue. Equilibrium dialysis experiments yielded the same quantitative data on the two classes of binding sites but revealed an additional type of binding at high concentrations of manganese not observable by the proton relaxation rate technique. The behavior of synthetic polynucleotides differed considerably from tRNA and could not serve as models for the different classes of binding sites of tRNA. Unlike tRNA, the homopolymers, poly A, I, C and U revealed only one class of binding sites which were independent of each other, and the value of the enhancement factor varied greatly with ionic strength. For an equimolar mixture of poly A + poly U and for the random copolymers (A,U) and (U,A), the value of ε{lunate}b was intermediate between poly A and poly U; the behavior of these systems varied drastically with concentration in a complex manner. Other ribonucleic acids, ribosomal RNA of E. coli and turnip yellow mosaic virus RNA resembled tRNA insofar as they had two classes of binding sites and the binding within the class of strong sites was co-operative. However, each had a characteristic number of sites and enhancement factor different from tRNA; for the ribosomal RNA and viral RNA, n1 was 0.095 and 0.150 per phosphate residue, respectively, and ε{lunate}b1 was 11.4 and 7.9, respectively. © 1969.