Delineation of Structural Domains in Eukaryotic 5S rRNA with a Rhodium Probe
The three-dimensional folding of Xenopus oocyte SS rRNA has been examined using the coordination complex Rh(phen)_2phi^(3+) (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)_2phi^(3+) binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the SS rRNA. On the wild-type SS rRNA, strong cleavage is found at residues U73, A74, AIOI, and U102 in the E loop and USO and G81 in helix IV; additional sites are evident at A22 and AS6 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full SS RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)_2phi^(3+) do, however, indicate that the apposition of several noncanonical bases as well as stem-loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured. Stem-loop junctions, helix bulges containing more than one unmatched nucleotide, and a U-U mismatch also appear to provide open major grooves for targeting by Rh(phen)_2phi^(3+). These distinctive structures may also be utilized for specific recognition by proteins, such as the transcription factor TFIIIA, that bind to 5S rRNA.
© 1992 American Chemical Society. Received November 6, 1991; Revised Manuscript Received January 14, 1992. Publication Date: April 1992. We are grateful for the financial support of the NIH (GM33309 to J.K.B.; GM38200 to P.W.H.). In addition, C.S.C. thanks the Parsons Foundation for their fellowship support.