The structure and dynamics of the polyadenylation inhibition element (PIE) RNA, free and bound to the U1A protein, have been examined using time-resolved FRET and 2-aminopurine (2AP) fluorescence. This regulatory RNA, located at the 3′ end of the U1A pre-mRNA, adopts a U-shaped structure, with binding sites for a single U1A protein at each bend (box 1 and box 2). The distance between the termini of the arms of the RNA is sensitive to its three-dimensional structure. Using Cy3/ Cy5 FRET efficiency to monitor binding of Mg 2+, we show that the PIE RNA binds two Mg2+ ions, which results in a restriction of its distance distribution of conformations. Local RNA structure probing using 2AP fluorescence shows that the structure of box 2 changes in response to Mg2+ binding, thus tentatively locating the ion binding sites. Steady-state FRET data show that the distance 〈R〉 between the termini of the PIE RNA stems decreases from 66 Å in the free RNA, to 58 Å when N-terminal RNA binding domains (RBD1) of U1A are bound, and to 53 Å when U1A proteins bind. However, anisotropy measurements indicate that both Cy3 and Cy5 stack on the ends of the RNA. To examine the consequences of the restricted motion of the fluorophores, FRET data are analyzed using two different models of motion and then compared to analogous data from the Cy3/fluorescein FRET pair. We conclude that the error introduced into distance calculations by stacking of the dyes is within the error of our measurements. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements; the mean distance is dependent on protein binding, but the breadth of the distributions indicates that the RNA retains structural heterogeneity.