A preorganized active site in the crystal structure of the Tetrahymena ribozyme.

Group I introns possess a single active site that catalyzes the two sequential reactions of self-splicing. An RNA comprising the two domains of the Tetrahymena thermophila group I intron catalytic core retains activity, and the 5.0 angstrom crystal structure of this 247-nucleotide ribozyme is now described. Close packing of the two domains forms a shallow cleft capable of binding the short helix that contains the 5' splice site. The helix that provides the binding site for the guanosine substrate deviates significantly from A-form geometry, providing a tight binding pocket. The binding pockets for both the 5' splice site helix and guanosine are formed and oriented in the absence of these substrates. Thus, this large ribozyme is largely preorganized for catalysis, much like a globular protein enzyme.

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.

Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron.

Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.

X-ray crystallography of large RNAs: heavy-atom derivatives by RNA engineering.

For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.