Visualization of a tertiary structural domain of the Tetrahymena group I intron by electron microscopy

Abstract

The P4-P6 domain RNA of the group I intron of Tetrahymena thermophila has previously been shown by chemical probing to be an independently folding domain of the intron's tertiary structure. To directly visualize this tertiary structure, the P4-P6 domain and two folding defective mutants were prepared for high-resolution electron microscopy using tungsten shadowcasting. In the presence of Mg2+, the P4-P6 domain predominantly consists of compact molecules, while the two mutant RNAs are nearly all rod-like molecules. The measured length of the rod-like molecules is 64 (+/- 6) bp, which agrees closely with the length expected for molecules containing secondary structure only. In the absence of Mg2+, the P4-P6 domain contains threefold or tenfold fewer compact structures (depending on the mounting procedures) than in the presence of Mg2+. These results provide direct evidence for the overall shape of the tertiary structure proposed on the basis of biochemical experiment, and they confirm the Mg2+ dependence of tertiary folding. An equilibrium between the extended (rod-like) and the compact structures is suggested, with the concentration of bound Mg2+ and different mounting methods influencing the direction of the equilibrium. The entire group I ribozyme (L-21 Sca I RNA) was also examined by electron microscopy in the presence of Mg2+, and was revealed to have a compact shape. These studies present a direct demonstration of long-range interactions in a catalytic RNA molecule.