Abstract

A striking and widespread observation is that higher-order folding for many RNAs is very slow, often requiring minutes. In some cases, slow folding reflects the need to disrupt stable, but incorrect, interactions. However, a molecular explanation for slow folding in most RNAs is unknown. The specificity domain of the Bacillus subtilis RNase P ribozyme undergoes a rate-limiting folding step on the minute time-scale. This RNA also contains a C2'-endo nucleotide at A130 that exhibits extremely slow local conformational dynamics. This nucleotide is evolutionarily conserved and essential for tRNA recognition by RNase P. Here we show that deleting this single nucleotide accelerates folding by an order of magnitude even though this mutation does not change the global fold of the RNA. These results demonstrate that formation of a single stacking interaction at a C2'-endo nucleotide comprises the rate-determining step for folding an entire 154 nucleotide RNA. C2'-endo nucleotides exhibit slow local dynamics in structures spanning isolated helices to complex tertiary interactions. Because the motif is both simple and ubiquitous, C2'-endo nucleotides may function as molecular timers in many RNA folding and ligand recognition reactions.