The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 174

phylogenetically conserved base pairs that are essential for the formation of
catalytic core, whereas the P3-P4 domains are not required for self-cleavage
but their presence enhances activity, especially at low magnesium ion
(Mg 2 þ ) concentrations. Biochemical analyses have identified a region 3 0
to these segments that enhance catalytic activity through additional pseu-
doknot formation. 25 The “catalytic core” of the glmS ribozyme was inves-
tigated by mutational analyses of key nucleotides and examination of highly
conserved nucleotide identities across bacterial species. The glmS ribozyme
was shown to contain an intricate, multi-strand core that utilized pseu-
doknot formation to correctly fold the RNA for subsequent interaction with
its coenzyme. 26 As detailed here, pseudoknot formation within and outside
the catalytic core seems to play an important role in glmS structure and
function.
Prior to the crystallization of glmS ribozyme, nucleotide analog inter-
ferencemapping(NAIM)andnucleotideanaloginterferencesuppression
(NAIS) studies revealed backbone and nucleobase functional groups
essential for ligand-dependent ribozyme function. NAIM was performed
with the natural ligand GlcN6P and identified necessary functional
groups and potential sites of ligand and metal ion interaction. Experi-
ments performed with phosphorothioate analogs and the thiophilic diva-
lent metal ion manganese identified metal ion-binding sites clustered
within the catalytic core. A strong site of interference occurred at residue
C2, near the cleavage site of the glmS ribozyme, independent of the pres-
ence of the P4 domain. However, for the P2.1 region, strong pho-
sphorothioate effects were observedforC29,G30,andA31forthe
truncated P1-P3 ribozyme, while weak effects occurred at A28, G30,
and A31 for the full-length P1-P4 ribozyme ( Fig. 5.1 ). Additionally,
the P1-P3 ribozyme demonstrated weak phosphorothioate effects in
P2.2 at G3 and G7, while the P1-P4 ribozyme exhibited strong interfer-
ence at G3. In both P1-P3 and P1-P4 ribozymes, similar but not iden-
tical patterns of phosphorothioate effects reside within the P2.1 and P2.2
segments, and identified two metal ion-binding sites not directly associ-
ated with coenzyme binding. 27
Further, NAIM identified essential 2 0 -OH and nucleobase functionali-
ties. NAIM performed in the presence of Mg 2 þ and GlcN6P for the
P1-P3 and P1-P4 ribozymes identified identical strong 2 0 -deoxy interfer-
ences at A58 and U59 for both constructs. In P2.1, however, strong
2 0 -deoxy effects were observed at G27 and A28 for the P1-P3 ribozyme,
but at C29 and G30 for the P1-P4 ribozyme. NAIM using purine analogs