Quantifying the energetic interplay of RNA tertiary and secondary structure interactions

To understand the RNA-folding problem, we must know the extent to which RNA structure formation is hierarchical (tertiary folding of preformed seconda ry structure). Recently, nuclear magnetic resonance (NMR) spectroscopy was used to show that Mg2+-dependent tertiary interactions force secondary stru cture rearrangement in the 56-nt tP5abc RNA, a truncated subdomain of the T etrahymena group I intron. Here we combine mutagenesis with folding computa tions, nondenaturing gel electrophoresis, high-resolution NMR spectroscopy, and chemical-modification experiments to probe further the energetic inter play of tertiary and secondary interactions in tP5abc, Point mutations pred icted to destabilize the secondary structure of folded tP5abc greatly disru pt its Mg2+-dependent folding, as monitored by nondenaturing gels. Imino pr oton assignments and sequential NOE walks of the two-dimensional NMR spectr um of one of the tP5abc mutants confirm the predicted secondary structure, which does not change in the presence of Mg2+, In contrast to these data on tP5abc, the same point mutations in the context of the P4-P6 domain (of wh ich P5abc is a subdomain) shift the Mg2+ dependence of P4-P6 folding only m oderately, and dimethyl sulfate (DMS) modification experiments demonstrate that Mg2+ does cause secondary structure rearrangement of the P4-P6 mutants ' P5abc subdomains. Our data provide experimental support for two simple co nclusions: (1) Even single point mutations at bases involved only in second ary structure can be enough to tip the balance between RNA tertiary and sec ondary interactions. (2) Domain context must be considered in evaluating th e relative importance of tertiary and secondary contributions. This tertiar y/secondary interplay is likely relevant to the folding of many large RNA a nd to bimolecular snRNA-snRNA and snRNA-intron RNA interactions.