The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 176

of GlcN6P were performed. These results showed little structural change in
the RNA in response to the ligand, indicating preorganization of the ligand-
binding pocket. 28 Fluorescence Resonance Energy Transfer (FRET) ana-
lyses and footprinting with terbium(III) and RNase V1 also indicated no sig-
nificant changes in secondary and tertiary structure upon ligand binding. 16
RNA folding seems to be the rate-limiting step for the glmS ribozyme as
cleavage initiated with Mg 2 þ and GlcN6P is slow (3 min 1 ) compared to
self-cleavage initiated by the addition of GlcN6P to glmS RNA prefolded
in the presence of Mg 2 þ (72 min 1 ). 29
In contrast, NAIM analyses of full-length P1-P4 versus truncated P1-P3
glmS RNA identified small changes in essential nucleotide identities within
the catalytic core. P4 is thought to stabilize the catalytic core as its presence
enhances catalytic activity, and mutations that disrupt its tertiary structure
can be compensated for by increasing the Mg 2 þ concentration. 25 Biochem-
ical analyses of the glmS ribozyme indicate that theRNA is structurally adapt-
able, dependent on the presence of the P3 and P4 domains. In addition, the
catalytic core is relatively plastic despite being essential to ligand binding and
catalysis. 27 These types of analyses revealed a great deal about the structure and
function of the glmS ribozyme prior to crystallography studies. Furthermore,
the fact that they were performed in solution may better indicate physio-
logically important features of ligand binding and catalysis by the glmS RNA.
6. glmS RIBOZYME STRUCTURE AND FUNCTION —
CRYSTALLOGRAPHIC ANALYSIS
A number of crystal structures of the glmS ribozyme have been solved
and have provided atomic level detail of this coenzyme-dependent RNA
catalyst. One of the first crystal structures solved was that of the glmS pre-
cleaved ribozyme in the presence or absence of the competitive inhibitor
glucose-6-phosphate (Glc6P). This structure revealed a rigid RNA pref-
ormed in presumably active, coenzyme-binding competent state, similar
to that proposed from biochemical assays. 12,28 The RNA exhibited a double
pseudoknot structure that is nearly identical to that of the postcleavage state
structure or to a structure determined with the natural ligand GlcN6P that
included a 2 0 - O -methyl modification at the cleavage site preventing catal-
ysis. 12-15,17 When crystallized with GlcN6P, interactions of the glmS
RNA with the ligand were found to involve hydrated Mg 2 þ coordinated
to the ligand phosphate. 13 Distinct among the binding interactions seen
for other riboswitches and their ligands, the glmS coenzyme-binding site