Crystal structure of a self-splicing group I intron with both exons

Abstract

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.

Probing the Role of a Secondary Structure Element at the 5‘- and 3‘-Splice Sites in Group I Intron Self-Splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G·U Pair in Self-Splicing

“…Consistent with these biochemical and structural results on model duplexes and the extended P1 duplex in the Tetrahymena ribozyme the recent X-ray crystal structures of the group I intron from Azoarcus also shows disrupted helical stacking at the G•U wobble pair (65,71). The potential importance of partial unstacking of the extended P1 duplex for self-splicing is discussed in the Summary and Implications section.…”

“…This structural picture is supported by the recent x-ray structures of the group I intron (65,71). Furthermore, this structural picture of internal G•U pairs is reflected in their energetics; substitution of a Watson Crick pair with a G•U pair in a model duplex has a larger effect within a helix than at the end (see Formation of the Extended P1 Duplex above) and an even larger effect of >2.8 kcal/mol measured on the ribozyme.…”

“…In the recent structure of the Azoarcus intron with P10 formed the distance between P9.0 and P10 (which contains both the IGS extension and the substrate-tail) exceeds 8 Å, too far to explain steric interference via a direct effect (65). It is possible that these distances are different in the Tetrahymena and Azoarcus RNAs or in different conformations of the ribozyme.…”

“…The simplest interpretation from these data is that there are no significant interactions between the P1 extension and the ribozyme. The recent crystal structure of the Azoarcus intron suggests the formation of a single hydrogen bond between the 2′-OH group of the last residue in J4/5 (G116 in Tetrahymena) and the 2′-OH group of the first residue of the IGS extension (G21 in Tetrahymena) (65). A previous measurement of the effect of removing tertiary interactions involving 2′-OH groups (by modification to 2′-H) gave effects of about 0.5 kcal/substitution, corresponding to an effect of about two-fold (75).…”

RNA takes center stage

“…The mechanism of some RNA enzymes is understood as well as or some may say in even greater detail than that of many protein enzymes. [16][17][18][19] In addition, we now have atomic resolution structures of many RNA molecules, [20][21][22][23][24][25][26][27][28] including the ribosome. [29][30][31][32] Such information has allowed us to make significant progress in determining the details of the cellular role of many RNA molecules, including the cell's largest RNA machine, the ribosome.…”