Metal-binding sites in the major groove of a large ribozyme domain

Abstract

Background: Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 A crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail.

Results: Heavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain.

Conclusions: Bound at specific sites in the P4-P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.