Structural Principles of RNA Catalysis in a 2′–5′ Lariat-Forming Ribozyme

RNA-catalyzed
lariat formation is present in both eukaryotes and
prokaryotes. To date we lack structural insights into the catalytic
mechanism of lariat-forming ribozymes. Here, we study an artificial
2′–5′ AG1 lariat-forming ribozyme that shares
the sequence specificity of lariat formation with the pre-mRNA splicing
reaction. Using NMR, we solve the structure of the inactive state
of the ribozyme in the absence of magnesium. The reaction center 5′-guanosine
appears to be part of a helix with an exceptionally widened major
groove, while the lariat-forming A48 is looped out at the apex of
a pseudoknot. The model of the active state built by mutational analysis,
molecular modeling, and small-angle X-ray scattering suggests that
A48 is recognized by a conserved adenosine, juxtaposed to the 5′-guanosine
in one base-pair step distance, while the G1-N7 coordinates a magnesium
ion essential for the activation of the nucleophile. Our findings
offer implications for lariat formation in RNA enzymes including the
mechanism of the recognition of the branch-site adenosine.