BIOINORGANIC CHEMISTRY A Short Course Second Edition

MAGNESIUM AND CATALYTIC RNA 293

5 ′ – 5 ′ - connected base on stem I formed a Watson – Crick base pair with C 10.4
creating a tethered hammerhead RNA in the crystal. Although the crystal
diffraction did not improve measurably, the new construct did help address
other criticisms of previous structures, namely that further movement and
conformational change was needed to visualize a possible transition state or
that modifi cations to crystal structure hammerheads prevented achievement
of a probable transition state. See discussion of the “ later intermediate ” struc-
ture (PDB: 379D) above in which a talo - 5 ′ - C - methyl modifi ed RNA (on A 1.1 ,
at the C17/A1.1 cleavage site) had been used to prevent cleavage in the crystal.
In the latter case of modifi cation, criticisms included the prediction that the
crystal structures visualized an “ off pathway ” conformational change not
related to achieving the actual hammerhead cleavage site transition state
structure. In solution, the PDB: 1NYI - modifi ed hammerhead construct (see
Figure 6.20 ) cleaved in a manner similar to that of the hammerhead initial
structures (PDB: 1MME and 299D) (see Figure 6.10 ), indicating that tethering
of stem I to stem II was negligible in solution.
However, tethering of stem I to stem II through C – G hydrogen bonding in
the crystal prevented cleavage completely in a construct where, importantly,
the RNA leaving group had not been modifi ed. Furthermore, the positions of
the C 17 nucleotide in the PDB: 1NYI structure was almost completely super-
imposable on that found for thetalo - 5 ′ - C - methyl modifi ed RNA of PDB: 379D,
allowing the argument that both structures were possible intermediates on the
correct pathway to a hammerhead cleavage - site transition state conformation.
The reference 58 authors argue that the phosphodiester tether that forms

Figure 6.20 Secondary structure of the tethered hammerhead ribozyme RNA 6 used
for X - ray crystallographic structures deposited in the Protein Data Bank as PDB: 1NYI
and 1Q29.

A

U

U

G A

A

A

A U

C

G

17

2.1

5 4

6 3

Catalytic Pocket

See key to figures 6.10 and 6.11.

G

12

13

14

15.1

9

8

7

scissile bond

Augmented

Stem II

Helix

C

C

C G

G C

11.3

11.2

G 11.1

10.4

10.3

10.2

10.1

5'

3'

Stem III

16.1

Stem I

1.1

3'

5'

U

C

A

C

C

A

C

U

G

G

G 1.5

1.4

1.3

1.2

2.5

2.4

2.3

2.2

C

U

C

A U

A

A

G

G

G

15.3

15.4

16.2 15.2

16.4

16.3

5'

G

3'

Stem II