MAGNESIUM AND CATALYTIC RNA 249
However, hydrogen - bonding characteristics could not be defi ned, nor could
magnesium ions be placed with confi dence. In the PDB: 1GRZ structure, the
P4 – P6 domain appears largely unchanged from that seen in the previous
crystal structure, PDB: 1GID, an encouraging note for researchers determining
structures of large RNA molecules. As before, the P4 – P6 domain is hairpin -
shaped with two coaxially stacked helical regions separated by a sharp bend.
The P3 – P9 domain, which had not previously crystallized, was found to be
wrapped around the P4 – P6 domain. Many interdomain interactions between
P4 – P6 and P3 – P9 were found, including at the catalytic active site. The P3 – P9
domain consists of fi ve helices, four of which (P8, P3, P7, and P9.0) are coaxi-
ally stacked. The P9 helix makes an approximate 90 ° bend and interacts with
the minor groove face of P5 in the P4 – P6 domain. The large size of the
intron —∼ 110 × 65 Å — is compared with that of the restriction endonuclease
Eco RI, which measures ∼ 40 to 60 Å in all directions. The researchers suggest
that the size difference indicates that a more effi cient active site may be built
with amino acids than with nucleotides. This last statement brings questions
of evolutionary biology into play as RNA catalysis is thought to have preceded
catalysis by amino acid - containing proteins and enzymes. Unfortunately, there
is neither time nor space to discuss this fascinating topic here.
Previous experimental results indicate that the secondary structures of
group I introns show a junction between the P4 – P6 and P3 – P9 domains that
involves the close approach of the P4, P6, P3, and P7 helices. Figure 4 of refer-
ence 15 shows the ribozyme catalytic core being formed in the PDB: 1GRZ
structure by the junction of these four helices: P3 (orange), P4 (yellow), P6
(pink), and P7 (green). Assembly of the helices into a catalytic conformation
involves base triples formed in J3/4 and J6/7. For instance, J3/4 contains three
bases: A104, A105, and A106. The nucleotide A104 stacks directly onto the P3
helix and appears to form an interacting quartet with nucleotides A270 (mis-
matched with A104), C217 (in P6), and A256 (mismatched with C217 in P6).
Base triples forming in the J6/7 region also help to orient the P7 helix.
Tetraloops are secondary structure domains that often mediate tertiary
interactions in RNAs. In the PDB: 1GRZ structure, a standard GAAA
tetraloop, L9 residues G323, A324, A325, A326, is modeled. This tetraloop
docks into the minor groove of P5 at base pair C203 – G118 and wobble base
pair U202 – G119.
The all - important exogenous guanosine - binding site (G site) of the intron,
G414 in the PDB: 1GRZ structure, is located within the P7 helix. An adjacent
A265 – U310 base pair has been implicated in guanosine binding, and residues
A306 (a strongly conserved nucleotide) and A261 are identifi ed as possibly
forming a “ gateway ” to the G site. Interactions within the P7 helix and back-
bone – backbone interactions with P4 in the vicinity of U106 and U107 distorts
this helix and compresses its major groove in such a way to provide a unique
binding site for the guanosine substrate (G414). The second group I intron
substrate, the 5 ′ splice site, located within the P1 duplex, is not present in the
PDB: 1GRZ crystallized ribozyme. However, the reference 15 authors do