The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 175

resulted in interference patterns indicative of contacts to the exocyclic amine
of A31, G30, and G57, and to the N7 imine of A28 and G30, whereas steric
restraints were implicated for A31 and A58.
Additional studies were performed using a unique variation of the NAIS
technique. Previously, NAIS has been employed to identify tertiary inter-
actions within other ribozymes, where analysis is based on the principle that
if a tertiary interaction is disrupted by site-specific deletion or alteration of
one functional group in an interacting pair, then no additional energetic
penalty will result from deletion or alteration of the second functional group.
For glmS studies, the typical NAIS methodology was modified by combin-
ing the use of a ligand analog (serving as a site-specific modification) with
interference mapping of the full-length glmS RNA. In this format, NAIS
can identify sites at which nucleotide analog incorporation that previously
caused interference with cognate ligand were tolerated with ligand analog.
NAIS experiments were performed using the ligand analog glucosamine
(GlcN), which lacks the 5 0 -phosphate functionality of GlcN6P but has been
previously demonstrated to stimulate P1-P4 glmS ribozyme activity at a
reduced rate. Thus, GlcN-dependent suppressions of interferences in the
glmS ribozyme reveal contacts between the phosphate moiety of GlcN6P
and functional groups within the RNA catalyst.
Within P2.2, GlcN-dependent suppression of phosphorothioate inter-
ference was observed at G3, but not at C2. For the P2.1 segment, suppres-
sion of phosphorothioate interference was observed at A28 and G30, but not
at A31. Therefore, NAIS data suggest that three glmS ribozyme phosphate
oxygens compose a portion of the ligand-binding site responsible for recog-
nition of the phosphate moiety of GlcN6P. Furthermore, NAIS performed
in the presence of Mg 2 þ and GlcN for the P1-P4 ribozyme demonstrated a
site of 2 0 -deoxy interference suppression at G30, essential imine (A28) and
amine (A31) groups, and steric boundaries within the phosphate recognition
site (identified by NAIS at A58 and A31 using diaminopurine as nucleotide
analog). The composition of the ligand-binding site indicates that GlcN6P
binds adjacent to the cleavage site within the catalytic core comprised of
interactions among the highly conserved sequence segments within P2.1
and P2.2.
NAIM and NAIS data aided in visualizing how the ligand was recog-
nized by the RNA and introduced a possible role for ligand in the mecha-
nism of self-cleavage as the ligand-binding site was found to lie close to the
cleavage site. 27 . To further investigate the ribozyme active site, hydroxyl
radical footprinting and crosslinking analyses in the absence and presence