BIOINORGANIC CHEMISTRY A Short Course Second Edition

MAGNESIUM AND CATALYTIC RNA 273

whether catalytic activity can be recovered (rescued). In 1996, the group pub-
lished a paper inPNAS describing the results when abasic nucleotides were
substituted at 13 critical positions on the hammerhead HH16 construct.^41
Figure 6.13 shows the 13 substituted positions, each marked with a dashed
outline. Figure 6.14 shows the structure of 7 - deazaguanine, a reduced abasic
nucleotide and a phenyl - substituted nucleotide.
For the 13 hammerhead HH16 mutants prepared by the reference 41
researchers, cleavage activity was signifi cantly reduced in all cases. In four
cases, at positions C 3 , A 9 , G 10.1 , and A 13 , addition of the correct base (U, A, G,
or C) rescued the activity of the ribozyme. A9X, for instance, is a ribozyme
where the adenine base at position 9 has been removed to generate an abasic
nucleotide (X = a nucleotide in which H substitutes for the removed base — see
Figure 6.14 ). This altered ribozyme exhibited a 4 × 10 − 4 times lower catalytic
rate than that for the wild - type ribozyme ( kk krel==abasic 22 /.wt 0 0004). When
exogenous adenine (3 mM) was added to the reaction mixture, catalysis was
rescued: k 2 obsA= 012 .min−^1 (or 300 - fold) compared to 1.1 ± 0.2 min − 1 for wild -
type ribozyme. For another variant, the G 10.1 X, where X represents the abasic
nucleotide mutant, the rescue was to wild - type behavior (from 0.04 min − 1 to
1.2 min − 1 ). The ribozyme positions C3X and A13X also exhibited rescue behav-
ior. The reference 41 authors put forth the following hypotheses: (1) Removal

Figure 6.13 Structure of the hammerhead ribozyme HH16. Dashed boxes surround
the 13 substituted positions.

C

C

A

C

A

C

C

C

A A

A

C

A

U

U

G

U

C

U A

A

A

A

C

G C

G C

C G

G A

A

C

G

U

C

U

U

C

C

G

U

G

G

6

17

1.5

1.4

1.3

1.2

1.1

2.5

2.4

2.3

2.2

2.1

4

5

3

Domain I

Domain II

G

G

G

G

G

G

G

G

G

11.4

11.3

11.2

12

13

14

11.1 G

15.1

15.3

15.4

15.2

10.4

10.3

10.2

10.1

9

8

7

16.2

16.4

16.3

scissile bond

16.1

5'

3'

stem (helix) I

stem (helix) II

L2.4

L2.3 L2.2

L2.1

stem (helix) III

3' 5'