bi062169g_si_002.pdf (104.04 kB)
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†
journal contribution
posted on 2007-04-24, 00:00 authored by Katrin Karbstein, Jihee Lee, Daniel HerschlagSeveral ribozyme constructs have been used to dissect aspects of the group I self-splicing
reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a
reaction analogous to the first step of self-splicing, in which a 5‘-splice site analogue (S) and guanosine
(G) are converted into a 5‘-exon analogue (P) and GA. This ribozyme preserves the active site but lacks
a short 5‘-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1
extension and P10 helix. The P1 extension forms at the 5‘-splice site in the first step of self-splicing, and
P10 forms at the 3‘-splice site in the second step of self-splicing. To dissect the contributions from the
IGS extension and the helices it forms, we have investigated the effects of each of these elements at each
reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS
extension, and with 5‘- and 3‘-splice site analogues that differ in their ability to form the helices. The
presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are
formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues
that do not form additional base pairs with the IGS extension. Analysis of a U·U pair immediately 3‘ to
the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the
L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary
structure specificity. Comparisons of the affinities of 5‘-splice site analogues that form only a subset of
base pairs reveal that inclusion of the conserved G·U base pair at the cleavage site of group I introns
destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson−Crick base
pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect
can be attributed to partial unstacking of the P1 extension at the G·U step. These results suggest a previously
unrecognized role of the G·U wobble pair in self-splicing: breaking cooperativity in base pair formation
between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10
after the first chemical step of self-splicing and release of the ligated exons after the second step of
self-splicing.