13C relaxation and dynamics of the purine bases in the iron responsive element RNA hairpin

Abstract

The iron responsive element (IRE) RNA hairpin contains a conserved six-nucleotide loop. The NMR structure of this loop showed that the positions of four of its bases are not tightly constrained, while the remaining two are hydrogen-bonded [Laing, L. G., and Hall, K. B. (1996) Biochemistry 35, 13586]. To investigate the flexibility of the RNA in the loop and in the stem, 13C NMR relaxation methods have been used to describe the dynamics of the purine bases. IRE hairpins containing [13C]guanosine and [13C]adenosine are used in NMR experiments to measure T1, T1rho, and NOE values of the bases as a function of temperature (20-37 degreesC). Data are analyzed using the Lipari-Szabo model-free formalism [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546] to determine order parameters and time scales of the motion. Results indicate that the purine bases in the stem have order parameters that are independent of temperature, although they show evidence of both fast (6-40 ps) motions and slower motions at 37 degreesC. The three purines in the loop exhibit increasingly complex motions with long (nanoseconds) correlation times as the temperature increases, suggesting that the loop structure has become disordered.