Recognition of Tertiary Structure in tRNAs by Rh(phen)₂phi³⁺, a New Reagent for RNA Structure-Function Mapping

With photoactivation Rh(phen)₂phi³⁺ promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)₂phi³⁺ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA^Phe and tRNA^Asp from yeast, the unmodified yeast tRNA^Phe transcript, and a chemically modified tRNA^Phe, as well as on a series of tRNA^Phe mutants. On tRNA^Phe strong cleavage is observed at residues G22, G45, U47, Ψ55, and U59; weaker cleavage is observed at A44, m⁷G46, and C48. On tRNA^Asp cleavage is found at residues A21 through G26, Ψ32, and U48, with minor cleavage apparent at A44, G45, A46, Ψ55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA^Phe transcript resembles closely that found on native yeast tRNA^Phe, but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA^Phe illustrate Rh(phen)₂phi³⁺ as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh-(phen)₂phi³⁺ appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bonded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m⁷G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex. The cleavage results are consistent with the notion that Rh(phen)₂phi³⁺ preferentially targets regions of tertiary structure in the tRNA because these regions are structured so that the major grooves are open and accessible to stacking by the complex. Since sites cleaved by the rhodium complex mark a range of tertiary structures, Rh(phen)₂phi³⁺ appears to be a powerful and unique probe in characterizing the folded structures of RNAs.