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As part of an effort to structurally characterize the complete  Neurospora  VS ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III–IV–V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS ribozyme II–III–VI three-way junction and its adjoining stems. In addition, we localize Mg 2+ -binding sites within this structure using Mn 2+ -induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a  cis  WC/WC G•A base pair, two base triples and two Mg 2+  ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge–bulge interaction and Mg 2+  ions are critical for the stable folding of the II–III–VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS ribozyme, but not with biophysical studies performed with a minimal II–III–VI junction that does not contain the II–VI bulge–bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique ribozyme.

The NMR structure of the II–III–VI three-way junction from the <i>Neurospora</i> VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure

The NMR structure of the II–III–VI three-way junction from the Neurospora VS ribozyme reveals a critical tertiary interaction and provides new insights into the global ribozyme structure