Communication between RNA folding domains revealed by folding of circularly permuted Ribozymes

JOURNAL OF MOLECULAR BIOLOGY

卷:373

Article

Lease, Richard A. ; Adilakshmi, Tadepalli ; Heilman-Miller, Susan ; Woodson, Sarah A.

关键词: circular permutation;    group I ribozyme;    folding kinetics;    hydroxyl radical footprinting;    kinetic partitioning;   

DOI  :  10.1016/j.jmb.2007.07.007

来源: Elsevier

【 摘 要 】

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, 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. (C) 2007 Elsevier Ltd. All rights reserved.

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