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three domains of U6 are involved in the formation of the active site in both
steps of splicing. Mutagenesis studies have suggested a critical role for the
three functionally crucial regions mentioned above in both the first and sec-
ond steps of splicing, although certain positions in these sequences seem to
be especially critical for the second step of splicing to occur.
15,17,42-47
Fur-
ther, as mentioned above, the hydroxyl radical footprinting experiments
performed on activated spliceosomes before and after the first step of splic-
ing
56
suggest that the remodeling of the snRNA structures between the two
steps is rather minor. Based on these data, it is likely that the main confor-
mational changes involve the removal of the first step products and position-
ing of the second step substrates in the active site, with minor changes in the
positioning of the snRNAs. Consistent with this possibility, mutational
complementation analyses in yeast have indicated that while strengthening
the 5
0
splice site-ACAGAGA interaction by increasing the base-pairing
potential allowed the first step of splicing to go to completion, it significantly
reduced the efficiency of the second step.
49
This finding not only confirmed
the previous data indicating that the first step of splicing occurs in immediate
proximity of the ACAGAGA box,
48,61
it also showed that the removal of
the first step products from the vicinity of this critical region of U6 is impor-
tant and likely required for the second step of splicing to occur. In summary,
current data suggest that U6 snRNA, similar to what has been observed for
analogous sequences in group II introns,
62,63
is likely to be involved in catal-
ysis of both steps of splicing. Although the active site elements likely undergo
a minor remodeling between the two steps of splicing, several other RNA-
RNA interactions such as the snRNA-substrate interactions seem to go
through significant
rearrangements during the catalytic cycle of
the
spliceosome.
58-60,62,63
5. AN RNA-CENTRIC VIEW OF THE ACTIVE SITE
Identity of the spliceosomal catalytic domains and the extent of
involvement of the RNA and protein components of the spliceosome in
catalysis have been a central and long-standing question in the splicing field.
If as suggested by the current data, U6 and U2 snRNAs are indeed the cat-
alytic components of the spliceosome, then certain residues in these snRNAs
should form the active site of the splicing reactions. Such residues are
expected to be first highly conserved and sensitive to mutations, and they
should also exhibit spatial proximity to the reacting groups of the splicing
reaction in spliceosomes undergoing the two catalytic steps of splicing. As
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