Background: Group I self-splicing introns catalyze sequential transest
erification reactions within an RNA transcript to produce the correctl
y spliced product. Often several hundred nucleotides in size, these ri
bozymes fold into specific three-dimensional structures that confer ac
tivity. The 2.8 Angstrom crystal structure of a central component of t
he Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 do
main, provides the first detailed view of metal binding in an RNA larg
e enough to exhibit side-by-side helical packing. The long-range conta
cts and bound ligands that stabilize this fold can now be examined in
detail. Results: Heavy-atom derivatives used for the structure determi
nation reveal characteristics of some of the metal-binding sites in th
e P4-P6 domain. Although long-range RNA-RNA contacts within the molecu
le primarily involve the minor groove, osmium hexammine binds at three
locations in the major groove. All three sites involve G and U nucleo
tides exclusively; two are formed by G U wobble base pairs. In the nat
ive RNA, two of the sites are occupied by fully-hydrated magnesium ion
s. Samarium binds specifically to the RNA by displacing a magnesium io
n in a region critical to the folding of the entire domain. Conclusion
s: Bound at specific sites in the P4-P6 domain RNA, osmium (III) hexam
mine produced the high-quality heavy-atom derivative used for structur
e determination. These sites can be engineered into other RNAs, provid
ing a rational means of obtaining heavy-atom derivatives with hexammin
e compounds. The features of the observed metal-binding sites expand t
he known repertoire of ligand-binding motifs in RNA, and suggest that
some of the conserved tandem G U base pairs in ribosomal RNAs are magn
esium-binding sites.