Compatible Structure of the VS Ribozyme

The length of stem-loop IV and the distal part of stem-loop VI can be varied to a great extent. The length of stem-loop V can be varied slightly (Lafontaine et al., 2002b), but the length of stem-loop III cannot be changed (Lafontaine et al., 2002b). There are no data available on stem-loop II. Furthermore, the A718 bulge in region III can be complemented (that is, replaced by a base pair, with the insertion of U after position 660) without significant loss of activity (Beattie et al., 1995; Lafontaine et al., 2002b). These changes constitute the basis of

Table 3.3 Enzymatic activities of different structures of the Neurospora VS ribozyme

Structure

"structure

Reference

Original structure

1.000

Deletion of the A652 bulge

0.013

Beattie et al.,

1995

Deletion of the A718 bulge

0.136

Lafontaine et

al.,

2002b

Pairing of the A718 bulge

0.820

Lafontaine et

al.,

2002b

Length 8 stem III

0.087

Lafontaine et

al.,

2002b

Length 7 stem V

0.045

Lafontaine et

al.,

2002b

Length 6 stem IV

0.470

Lafontaine et

al.,

2002b

Length 4 stem IV

0.590

Lafontaine et

al.,

2002b

Length 8 stem VI

0.970

Lafontaine et

al.,

2001b

Length 6 stem VI

1.000

Lafontaine et

al.,

2001b

other acceptable structures beside the wild-type structure. Table 3.3 lists various structures and their activities.

In order to maintain the wild-type total length N = 144 constant, all structures including deletions or insertions were slightly modified in our study. Single-stranded regions were removed from or added to the beginning and the end of the structure in all possible combinations. We allowed some mispairs in the structure, but a mispair can only be considered if the following criteria are met (in what follows all numbers refer to positions in the wild-type sequence. In the alternative structures these positions change according to the shift due to the insertion or deletion):

• Two mispairs cannot be adjacent. There are six experimentally tested mutants that have mispairs at adjacent positions. Four of them have absolutely no activity ([G727C, U728A], [G722C, C723G], [G762C, C763G], [A759U, C760G]). In the case of the remaining two mutants ([G716C, U717A], [A661U, C662G]) the activities are 0.02 and 0.06, respectively.

• The pairs [653:771], [654:770], and [655:769] in stem-loop II have to be paired. The mispair mutants of the region ([G653C], [C771G], [G655C]) do not have measurable activities, except for the mutant [C769G] whose relative enzymatic activity is 0.18.

• The base pairs adjacent to a junction have to be paired. The stable forming of the junction is critical for enzymatic activity. If the bases next to a junction do not form a base pair, then the structure of the junction changes considerably and enzymatic activity would vanish. Accordingly, the following base pairs have to be paired: [658:721], [663:715], [666:685], [687:709], [722:763].

• A stem-loop cannot contain more than two mispairs. Generally, two mispairs in the same region greatly reduce activity (0.78 [G704C, U706A], 0.54 [U670A, C672G], 0.28 [A748U, U750A], 0.25 [A735U, U737], 0.07 [A690U, C692G], 0.06 [A661U, C662G], and 0.02 [G716C, U717A]) or abolish it completely (see the adjacent mispairs mentioned above or the mutant [G679C, A681U]). We assume that if a stem-loop contains more than two mispairs, then the resulting molecule would not have any enzymatic activity.

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