METAL-BINDING SITES IN THE MAJOR GROOVE OF A LARGE RIBOZYME DOMAIN

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.