Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 73

Table 2.12 Structural indexes characterizing the relative positions between C1.1 and G8
WT U7C G8I C3U/G8A C3U/G8D C3G/G8C

r 7 4.17 (0.38) 4.15 (0.35) 4.19 (0.36) 4.70 (0.69) 4.89 (0.49) 4.24 (0.33)

y 7 110.2 (11.3) 109.3 (9.6) 111.6 (9.8) 116.8 (13.1) 123.7 (9.0) 111.1 (8.1)
r 8 3.82 (0.34) 3.84 (0.37) 3.86 (0.36) 4.06 (0.45) 4.27 (0.50) 4.11 (0.43)

y 8 47.6 (8.5) 45.4 (8.3) 43.8 (7.3) 61.1 (20.8) 61.4 (11.9) 32.5 (9.0)

d-WT d-U7C d-G8I d-C3U/G8A d-C3U/G8D d-C3G/G8C

r 7 4.23 (0.32)

y 7 111.6 (7.1) 105.7 (7.9) 111.8 (8.7) 108.8 (7.9) 108.6 (10.5) 115.1 (9.1)
r 8

4.04 (0.25) 3.98 (0.25) 4.13 (0.34) 4.00 (0.26) 4.40 (0.36)

3.76 (0.31) 3.62 (0.29) 3.65 (0.34) 3.51 (0.27) 4.13 (0.38) 3.56 (0.27)

y 8 49.4 (6.6) 47.6 (6.1) 54.1 (7.3) 51.2 (6.9) 54.0 (7.0) 55.6 (7.3)

This table lists key structural indexes to characterize base stacking between G8 and C1.1 for different
mutants. Data analysis was performed over the last 65 ns of each simulation with a 10 ps sampling fre-
quency. Distance and angles ( Fig. 2.10 ) are in ˚ and degrees, respectively. r 7 is r (C1.1:N1, G8:N9); y 7 is y
(C1.1:C2, C1.1:N1, G8:N9); r 8 is r (C1.1:O4 0 , G8:C1 0 ); and the torsion angle y 8 is y (C1.1:C1 0 , C1.1:
O4 0 ,G8:C1 0 , G8:N9). SDs are listed in parentheses. Boldface font is used to highlight key quantities that
are significantly altered with respect to the WT simulation upon mutation and that are discussed in the
text. The notation “d-” denotes the activated precursor state simulations having the C17:O2 0 deprot-
onated. When G8 is mutated to a C, G8:N9 is replaced by C8:N1.

distance between the A9 and scissile phosphates ( d 0 ) increases by 2.67 ˚ rel-
ative to the WT simulation, breaking key hydrogen bonds between the O2 0
nucleophile of C17 and N1 of G12 (the implicated general base). These per-
turbations in the reactant state would prevent activation of the nucleophile
and progress toward the transition state.

5.1.2.2 G8I mutation is relatively benign
The G8I mutation does not significantly alter catalytic activity, where the
measured rate reduction is less than a factor of two. 94,96 The removal of
the exocyclic amine at the C2 position weakens the hydrogen-bonded base
pair with C3, but does not alter the structure ( Fig. 2.11 and Table 2.8 ).
None of the structural features derived from the simulations in either the
reactant state or activated precursor state show any marked differences from
the WT simulations. Overall, the marginal effect on catalysis is likely a
consequence of modest weakening, but not disruption of the base pair
between C3 and G8.