MAGNESIUM AND CATALYTIC RNA 275
In a 1998 RNA paper, the Herschlag group further examined base rescue
at four positions: C 3 , A 9 , G 12 , and A 13 probing transition state interactions at
these hammerhead positions.^42 Their model for base rescue, illustrated in
Figure 3 of reference 42 , shows that removal of a base interferes with the
ability of the hammerhead ribozyme core to achieve its catalytic structure but
that addition and binding of an exogenous base can allow an active catalytic
structure to be generated. This active structure may exhibit enhanced substrate
cleavage over that of the wild - type ribozyme. An equation for calculating the
observed cleavage rate is given by
k
kk
Kd
22
1obs rescueBase
Base=
+
+
⎛
⎝⎜
⎞
⎠⎟
[]
[]
(6.2)
wherek 2 is the cleavage rate constant in the absence of the rescuing base, Kd
is the dissociation constant of the base, and krescue is the apparent second - order
rate constant for the rescue = kK′ 2 / d.
In the model scheme, k′ 2 is the cleavage rate constant for the ternary ribo-
zyme – substrate – rescuing base complex. It was found that krescue depended on
all the interactions happening in the transition state when the added rescuing
base forms the ternary ribozyme – substrate – rescuing base complex from the
altered ribozyme – substrate precursor. Many pyrimidine and purine bases
were used to test the involvement of specifi c base functional groups in achiev-
ing catalysis.
The cytosine at position 3 was removed to form C3X and the catalytic rate
decreased 10^5 - fold. Addition of 50 mM cytosine provides a rescue of 20 - fold
for the C3X variant while addition of cytosine to the wild - type ribozyme –
substrate complex does not affect the catalytic rate. Purine bases and uracil
did not provide rescue for the C3X variant whereas isocytosine (see Figure
6.15 ) does provide effi cient rescue. The reference 42 authors concluded that
the same hydrogen bonding groups on the pairing face are necessary for rescue
because isocytosine and cytosine provide the same needed atoms in the correct
alignment (see bold atoms in Figure 6.15 ).
Figure 6.15 Important hydrogen - bonding positions for (A) cytosine and (B) isocyto-
sine in hammerhead ribozymes.
NNHNOH HN NNOHHHisocytosine3H bond to C 17 NH 2 group H bond to C 17 NH 2 groupABcytosine3Bold positions are important for transition state hydrogen bonding in hammerhead ribozymes.