The Role of Bulges and Hinges in RNA Folding

Abstract

Long-range tertiary interactions govern RNA structural assembly, which is a critical step toward RNA biological functionality. Thereby, a universal strategy has emerged for global conformational change; flexible junctions enable unpaired nucleotides to act as beacons between helical regions. Bulges, for example, are versatile secondary structural elements implicated in helix recognition and packing. The P4-P6 domain of the Tetrahymena ribozyme utilizes this folding strategy by hinging to form two inter-helical tertiary contacts, the adenine-rich (A-rich) bulge and the tetraloop-tetraloop receptor interactions. To explore the kinetic and thermodynamic properties of tertiary contact formation, we probe the P4-P6 domain hinging and ribose zippering that forms the A-rich bulge interaction using single-molecule FRET methods. We obtain the docking and undocking rates of the A-rich bulge and P4 helix as function of cation concentration and temperature. Docking is accelerated and undocking decelerated by Mg2+. In spite of rapid docking at high [Mg2+] (kdock = 20 ± 2 s−1), the A-rich bulge interaction is only marginally stable (Kdock = 1.2 ± 0.1). These results support that the role of the A-rich bulge is to kinetically direct P4-P6 domain folding while thermodynamic stability is added through the tetraloop-receptor interaction. Formation of the A-rich bulge contact shows specificity for divalent cations, with a preference for Mg2+ as anticipated from the Mg2+ coordination observed in structural data. A significant kinetic heterogeneity is characterized; only 50% of the molecules exhibit efficient folding at high [Mg2+]. Mutations of the A-rich bulge construct reveal a crucial role of the P4-P6 secondary architecture in enabling the A-rich bulge contact.