Folding intermediates of a self-splicing RNA: mispairing of the catalytic core.
The Tetrahymena thermophila self-splicing RNA is trapped in an inactive conformation during folding reactions at physiological temperatures. The structure of this metastable intermediate was probed by chemical modification interference and site-directed mutagenesis. In the inactive structure, an incorrect base-pairing, which we call Alt P3, displaces the P3 helix in the catalytic core of the intron. Mutations that stabilize Alt P3 increase the fraction of pre-rRNA that becomes trapped in the inactive structure, whereas mutations that destabilize Alt P3 reduce accumulation of this conformer. At high concentrations of Mg2+, the yield of correctly folded mutant pre-rRNAs is similar to wild-type RNA. Under these conditions, the rate of folding for mutant RNAs is slower than for the wild-type, but is increased by addition of urea. The results show that slow folding of the Tetrahymena pre-rRNA is a consequence of non-native secondary structure in the catalytic core of the intron, which is linked to an alternative hairpin in the 5' exon. This illustrates how kinetically stable, long-range interactions shape RNA folding pathways.
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742-2021, USA.
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Pan
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Woodson
D000818:Animals; D001665:Binding Sites / Q000235:genetics*; D007438:Introns / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics; D016808:Tetrahymena thermophila / Q000737:chemistry* / Q000235:genetics
D012322:RNA Precursors; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D008274:Magnesium