SHAPE analysis of long-range interactions reveals extensive and thermodynamically preferred misfolding in a fragile group I intron RNA.
Most functional RNAs require proteins to facilitate formation of their active structures. In the case of the yeast bI3 group I intron, splicing requires binding by two proteins, the intron-encoded bI3 maturase and the nuclear encoded Mrs1. Here, we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry coupled with analysis of point mutants to map long-range interactions in this RNA. This analysis reveals two critical features of the free RNA state. First, the catalytic intron is separated from the flanking exons via a stable anchoring helix. This anchoring helix creates an autonomous structural domain for the intron and functions to prevent misfolding with the flanking exons. Second, the thermodynamically most stable structure for the free RNA is not consistent with the catalytically active conformation as phylogenetically conserved elements form stable, non-native structures. These results highlight a fragile bI3 RNA for which binding by protein cofactors functions to promote extensive secondary structure rearrangements that are an obligatory prerequisite for forming the catalytically active tertiary structure.
Duncan
Caia D S
CD
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA. weeks@unc.edu
Weeks
Kevin M
KM
D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D007438:Introns; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D010802:Phylogeny; D021122:Protein Subunits; D012331:RNA, Fungal / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics
D021122:Protein Subunits; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D009713:Nucleotidyltransferases; C034038:mRNA maturase; D004722:Endoribonucleases