Communication Between RNA Folding Domains Revealed by Folding of Circularly Permuted Ribozymes

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 degrees 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 degrees 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 T(1) 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.

Compact Intermediates in RNA Folding

“…One permutant of the Tetrahymena ribozyme nicked a linker between the P4–P6 and P3–P9 domains. Their folding was totally decoupled, so that the stable P4–P6 domain folded even more rapidly than before, but the P3 pseudoknot and the ribozyme core folded much more slowly, diminishing the yield of active RNA (48). A similar decoupling between domains was obtained by permuting the sequence of RNase P ribozyme (60).…”

The Ordered Transcription of RNA Domains Is Not Essential for Ribosome Biogenesis in Escherichia coli

“…Circular permutation usually occurs to proteins during the course of molecular evolution (Lo et al, 2009). In RNA molecules, CPs of tRNAs and the group I ribozyme were artificially constructed to study RNA folding and RNA-RNA interactions (Lease et al, 2007;Pan et al, 1991). In ribosomes, mutant 50S subunits were reconstituted in vitro from a series of circular permutated 23S rRNAs with chemical modifications to probe the catalytic mechanism of the peptidyl-transfer reaction (Erlacher et al, 2005).…”

The 23S Ribosomal RNA From Pyrococcus furiosus Is Circularly Permuted

“…These structural differences may confer different biological properties to the circularly permuted molecular species. Circular permutation has been used as a method to study various properties of RNA molecules, e.g., the order of folding ( Pan, 2000 ; Lease et al, 2007 ), including the folding ( Kitahara and Suzuki, 2009 ) or tethering of rRNA ( Fried et al, 2015 ; Orelle et al, 2015 ) and to make molecular tools, e.g., permuted group I introns as vehicles for producing circularized exons ( Puttaraju and Been, 1992 ; Ford and Ares, 1994 ) or permuted rRNA in a protocol for the incorporation of non-natural nucleoside analogs ( Erlacher et al, 2011 ). In nature, circular permuted RNAs can be produced by RNA processing or by genomic rearrangement.…”