Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 80

(d-G5I). For the double mutant d-C3U/G8D, although all U3-D8 hydro-
gen bonds are maintained ( Table 2.9 ), the C1.1-G8 base stacking distance is
slightly elongated as indicated by larger r 7 and r 8 in Table 2.12 . As a result,
the general acid, G12:O2 0 is shifted away from the leaving group C1.1:O5 0
( r HA in Table 2.9 ).
5.2.3 C3/G8 mutations may induce structural changes that have
remote consequences at the general base step
There is evidence that mHHRmotifs are conformationally variable, and that
there is an equilibrium between active and inactive conformational
states. 84,149,152 It has been hypothesized that mHHR and eHHRmay utilize
a similar dynamic reaction mechanism for catalysis, and that observed cor-
relations between mutations at C17 and C3/G8 may be explained by tran-
sient pairing between residues 3 and 17 and 8 and 13 arising from
interactions in the inactive conformation. 149 This supposition was based
in part on the observation that C3/G8 are distant from C17 in the eHHR
structures, making it difficult to rationalize the observed correlations
between mutations at C17 and C3/G8 in an active conformational state,
as in the inactive mHHR structures, C3 pairs with C17 and G8 pairs with
A13. 2,72,87
Our simulation results of the active eHHR, on the other hand, suggest
that mutations at C3/G8 in some cases may induce remote changes in the
structure and dynamics at C17 that affect the general base step of the reac-
tion. Recent kinetics studies have indicated that the native eHHR con-
structs exhibit less conformational variability than the mHHR motifs, and
they maintain a higher population of active conformations. 84,152 Hence,
the present simulation results afford an alternative interpretation of experi-
mentally observed correlations between mutations at C17 and C3/G8 posi-
tions that do not rely, at least as heavily, on the assumption that mutants of
the eHHR are in a dynamic equilibrium that populate an inactive confor-
mation with contacts resembling that of the mHHR.
5.2.4 Variation of base stacking interactions of G8 and C3 with C1.1
and G2.1 may lead to new HHR motifs with modified activity
Our results suggest that the identities of nucleotides at C3 and G8, including
some double mutants that preserve base pairing, can adversely effect HHR
catalytic activity. This implies that the environmental effect due to base stac-
king interactions between G8/C1.1 and C3/G2.1 may be important to hold
G8/C1.1 in the proper relative position for the general acid step of HHR