The Role of snRNAs in Spliceosomal Catalysis - Progress in Molecular Biology and Translational Science - page 205

could clearly demonstrate a metal ion specificity switch, thus providing
definitive evidence for functionally critical inner sphere metal ion coordina-
tion by these two oxygens which are the leaving groups of the first and sec-
ond steps of splicing, respectively ( Fig. 6.4 ). Previous phosphorothioate
substitution studies had shown that replacing the pro-Rp nonbridging phos-
phate oxygen at the 5 0 splice site blocks the first step of splicing, a finding
which could be due to the involvement of this oxygen in coordinating func-
tionally important metal ions. 72,73 As mentioned above, crosslinking and
mutational complementation analyses have indicated that at the time of
catalysis of the first step of splicing, the 5 0 splice site is positioned in close
proximity to the ACAGAGA box, partially by base pairing to this
sequence. 17,48,49 This base-pairing interaction positions the 5 0 splice site
directly across the duplex from the phosphorothioate interference sites in
the ACAGAGA sequence ( Fig. 6.4 ). 17 Thus, it is possible that the metal
ion bound by the ACAGAGA sequence is the same one that is coordinated
by the nonbridging oxygen and the leaving group at the 5 0 splice site. It has
been proposed that this metal ion likely facilitates the departure of the leav-
ing oxyanion by stabilizing the developing negative charge, consistent with a
proposed catalytic strategy for ribozyme-catalyzed phosphoryl transfer

reactions. 74,75
Taken together, significant evidence indicates that the primary sequence
of U2 and U6 snRNAs is sufficient to induce their folding into a base-paired
structure that not only contains many of the long-range interactions
observed in the catalytically active spliceosomes, but also has the ability to
form binding pockets for functionally critical metal ions. Further, base-
pairing interactions between U6 and U2 snRNAs and with the 5 0 splice site
and the branch site play a significant role in positioning of the substrates in
the active site of the spliceosomes, 17 suggesting that the majority of RNA-
based interactions found in the spliceosomal active site can be reconstituted
in protein-free systems in vitro .

7. CATALYTIC ACTIVITY OF THE snRNAs IN ISOLATION
As mentioned previously, the in vitro -assembled, base-paired complex
of U2 and U6 snRNAs recapitulates to a large extent the structural features
and metal ion-binding properties of the U2-U6 complex formed at the
spliceosomal catalytic core, which in turn has close structural and functional
similarities with the catalytically crucial domains of group II introns. These
parallels point to the intriguing possibility that the folded structure of the