MAGNESIUM AND CATALYTIC RNA 241
position) for RNA as the substrate segment. These experiments teased out
some mechanistic details such as the following: (1) Tertiary interactions with
certain 2 ′ - OH groups are important for docking the substrate – internal guide
sequence, IGS, helix into the active site; (2) the rate of chemical cleavage step
is reduced 40,000 - fold, indicating that a lack of functional groups (2 ′ - OH for
one) that could protonate the leaving group is necessary for activity. Observa-
tions that magnesium ions facilitated both the stabilization of the leaving
group and the activation of the guanosine nucleophile in group I intron ribo-
zymes provided analogies to a number of protein metalloenzymes such as
phosphodiesterases and RNA and DNA polymerases.^12
Three - dimensional structures of ribozymes were highly desired at this
time, but in the early 1990s no RNA larger than tRNA had ever been crystal-
lized, let alone had its structure determined. The Tetrahymena ribozyme 160
nucleotide (nt) P4 – P6 domain proved amenable to crystallization, and in 1996
its structure was solved by isomorphous replacement of heavy atoms and
MAD (multiwavelength anomalous diffraction) phasing (PDB: 1GID).^13 See
Section 3.3.3 for a discussion of these X - ray crystallographic techniques. New
tertiary structure motifs and metal ion interactions in the relatively solvent -
inaccessible RNA core validated biochemical data that had been previously
determined, and much of the solvent phase biochemistry agreed with the solid -
phase crystal structure. More will be said about the Tetrahymena ribozyme
structure after this introduction. Next the so - called hammerhead ribozyme
structure was solved (PDB: 1HMH),^14 followed by the structure of a 247 - nt
catalytically active group I ribozyme revealing an active site much like
that found in a protein enzyme — that is, pre - organized for activity (PDB:
1GRZ).^15
In 2002, two particular areas of interest to the reference 1 author T. R. Cech
were: (1) interactions of proteins with ribozymes, as proteins enable or extend
RNA activity; and (2) solving structures of ribozymes at atomic resolution. As
will be seen in the following paragraphs, both of these areas are still being
addressed by researchers.
6.2.2 Analyzing the Role of the Metal Ion
An informative review article, written in 2003, forms the basis for much of
the following discussion.^16 Metal ions, monovalent and divalent, are known
cofactors in catalytic RNA (ribozyme) activity, and they are critical for
both their structural and catalytic properties. Most ribozymes are very sensi-
tive to ionic conditions, and all require divalent cations such as Mg 2+ for activ-
ity. Since RNA folding is highly dependent on electrostatic interactions, metal
ions are believed to be required for formation of correct structural conforma-
tions. Cations can also enable catalysis in the following ways: (1) as general
bases perhaps as metal - hydroxo species, activating the nucleophile; (2) as
general acids perhaps as metal - aqua species, aiding in the protonation of
the leaving group; and (3) as charge neutralizers, stabilizing transition states.