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Abstract: To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1–3 min at 42 °C. Permutation of the 5′ end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 °C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5–8 min). By contrast, circular permutation of the 5′ end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60–80% folded within 30 s. RNase T1 digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5′-to-3′ order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center. [Copyright &y& Elsevier]

Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5′ domain of the Escherichia coli 16S rRNA at 20ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20mM MgCl2, many tertiary interactions appeared within 20ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly. [Copyright &y& Elsevier]