MAGNESIUM AND CATALYTIC RNA 263
hammerhead ribozyme crystallographic studies. Domain 1 (or I) contains a
U - turn motif (similar in tertiary fold to the uridine turn observed in tRNA
structures) comprising the fi rst four residues 3 ′ to stem (helix) 1, these are
conserved residues C 3 , U 4 , G 5 and A 6. In addition, domain I includes the resi-
dues 3 ′ and 5 ′ to the cleavage site — that is, C 17.0 and X 1.1. Domain 2 (or II)
consists of parts of stems (helices) 2 and 3 that are coaxially stacked upon one
another and is composed of eight conserved residues. It contains a connection
between stems 2 and 3 that consists of the three noncanonical (non - Watson –
Crick) pairs (G 12 – A 9 , A 13 – G 8 and A 14 – U 7 ). The G - A/A - G mismatches are a
highly conserved motif in many ribosomal RNA secondary structures. A three -
way junction connects A 6 , U 7 , U 16.1 , and C 17. The overall shape resembles a Y
with stems 2 and 3 stacked, and stems 1 and 2 subtended by an acute angle.
(See Figures 6.10 , 6.11 , and 6.12 .)
Many researchers refer to stems 1, 2, and 3 using their Roman numeral
equivalents — that is, stems I, II, and III. These motifs are also denoted as
helices I, II, and III. It should be noted at the beginning of this hammerhead
ribozyme discussion that structure – function relationships, the role of various
nucleobases, metal ion participation in catalysis, and other features of the
system have not been completely delineated and in some cases remain con-
troversial. Globally, the hammerhead fold appears to be similar in both solu-
tion and solid - state studies. In solution, however, the central core of the
hammerhead construct appears to be highly dynamic. This may account for
different experimental results among the analytical techniques used in solu-
tion and certainly explains some distinct differences seen between solution
and solid - state (X - ray crystallographic) structures.
Several hammerhead secondary structural motifs (constructs) are shown in
Figures 6.10 , 6.11 , and 6.12. An arrow indicates the cleavage site at the C 17.0 – X 1.1
position in the substrate (X stands for nucleotide found at this position in the
different constructs). The numbering system follows that of Hertel et al.^34 and
has been applied to many different hammerhead constructs. The basic system
starts numbering at the nucleotide 3 ′ to the cleavage site — that is, X 1.1. The
decimal notation indicates that the nucleotide is part of a helix and is paired
with another nucleotide. In Figure 6.11 for instance, G 1.1 of the substrate strand
pairs with C 2.1 in the ribozyme strand. Seventeen nucleotides in the central
core are numbered in a clockwise fashion: G 1.1 , C 2.1 , C 3 , U 4 , G 5 , A 6 , U 7 , G 8 , A 9 ,
G10.1 , C 11.1 , G 12 , A 13 , A 14 , A 15.1 , U 16.1 , and C 17. Nucleotides in loops are numbered
by the helix they are contained in, for example: G L2.1 , A L2.2 , A L2.3 , A L2.4 , in Figure
6.11. In the hammerhead, cleavage occurs between nucleotides 17 and 1.1,
resulting in phosphate 1.1 attached to ribose 17 as a 2 ′ ,3 ′ cyclic phosphodiester
(see Figure 6.9 ). Mutations are denoted, for example, by G5A, where a guanine
residue at position 5 is changed to an adenine. Two methods for illustrating
the constructs are shown in Figures 6.11 and 6.12. The second emphasizes
nucleotide numbering in the central core. Bimolecular hammerhead (ribo-
zyme plus substrate) catalysis can be described by three principal steps: (1)
association of the ribozyme to the substrate strand; (2) the chemical cleavage