Evidence for preorganization of the glmS ribozyme ligand binding pocket.
We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern. Cross-linking with short-wave UV light of the complex yielded similar overall results. In addition, ribozyme-substrate complexes cross-linked in the absence of GlcN6P could be gel purified and then activated in the presence of ligand. One of these active cross-linked species links the base immediately 3' of the cleavage site to a highly conserved region of the ribozyme core and could be catalytically activated by ligand. Combined with recent studies that argue that GlcN6P acts as a coenzyme in the reaction, our data point to a riboswitch mechanism in which ligand binds to a prefolded active site pocket and assists in catalysis via a direct participation in the reaction chemistry, the local influence on the geometry of the active site constituents, or a combination of both mechanisms. This mode of action is different from that observed for other riboswitches characterized to date, which act by inducing secondary and tertiary structure changes.
Hampel
Ken J
KJ
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Drive, 222 Stafford Hall, Burlington, Vermont 05405, USA. khampel@uvm.edu
Tinsley
Melissa M
MM
D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D017665:Hydroxyl Radical / Q000737:chemistry; D008024:Ligands; D009690:Nucleic Acid Conformation; D023302:Protein Footprinting; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D014466:Ultraviolet Rays
D008024:Ligands; D016337:RNA, Catalytic; D017665:Hydroxyl Radical; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine