Christopher D. Downey1, José Hodak2, David Nesbitt2, Arthur Pardi

CHRISTOPHER D. DOWNEY; JOSÉ HODAK; DAVID NESBITT; ARTHUR PARDI

Bethesda Maryland 20814, USA

Conferencia; Conference of The Biophysical Society; 2004

The Biophysical Society

Structural and biochemical studies of RNA molecules indicate that the functional and active sites of RNAs often exhibit considerable conformational flexibility. There is currently only limited information about these RNA structural dynamics and how they contribute to RNA folding and function. Single molecule fluorescence spectroscopy has emerged as a powerful tool for resolving the equilibrium conformational changes in RNA molecules in real time. In this study we apply single molecule fluorescence to examine the structural dynamics of an important RNA tertiary contact, the GNRA tetraloop-11 n.t. receptor motif. The tetraloop specifically docks to the receptor in the presence of Mg2+. This interaction contributes to the proper folding and activity of a variety of structured RNAs, including the group I and II introns and RNaseP. Our study examines the rates of docking and undocking and the associated equilibrium for a GAAA tetraloop-11 n.t. receptor tertiary contact in the absence of other tertiary contacts. We find that the rate constant for docking of the motif increases 10-fold between zero and saturating Mg2+ conditions, whereas the corresponding change in rate constant for undocking is only 3-fold. The results indicate that the local structural elements necessary for docking are stabilized by Mg2+ and at least partially formed prior to formation of the tertiary contact. We also examine a variety of constructs, which have variations in the tetraloop-receptor sequences and in the sequence and length of the region linking the two domains, in order to probe how the molecular context of the tertiary contact affects the docking kinetics and equilibrium constant.