Structure and ion-dependent folding of k-junctions

Abstract

k-Junctions are elaborated forms of kink turns with an additional helix on the nonbulged strand, thus forming a three-way helical junction. Two were originally identified in the structures ofArabidopsisandEscherichia colithiamine pyrophosphate (TPP) riboswitches, and another called DUF-3268 was tentatively identified from sequence information. In this work we show that theArabidopsisandE. coliriboswitch k-junctions fold in response to the addition of magnesium or sodium ions, and that atomic mutations that should disrupt key hydrogen bonding interactions greatly impair folding. Using X-ray crystallography, we have determined the structure of the DUF-3268 RNA and thus confirmed that it is a k-junction. It also folds upon the addition of metal ions, though requiring a 40-fold lower concentration of either divalent or monovalent ions. The key difference between the DUF-3268 and riboswitch k-junctions is the lack of nucleotides inserted between G1b and A2b in the former. We show that this insertion is primarily responsible for the difference in folding properties. Finally, we show that the DUF-3268 can functionally substitute for the k-junction in theE. coliTPP riboswitch such that the chimera can bind the TPP ligand, although less avidly.