Examination of the structural and functional versatility of glmS ribozymes by using in vitro selection.
Self-cleaving ribozymes associated with the glmS genes of many Gram-positive bacteria are activated by binding to glucosamine-6-phosphate (GlcN6P). Representatives of the glmS ribozyme class function as metabolite-sensing riboswitches whose self-cleavage activities down-regulate the expression of GlmS enzymes that synthesizes GlcN6P. As with other riboswitches, natural glmS ribozyme isolates are highly specific for their target metabolite. Other small molecules closely related to GlcN6P, such as glucose-6-phosphate, cannot activate self-cleavage. We applied in vitro selection methods in an attempt to identify variants of a Bacillus cereus glmS ribozyme that expand the range of compounds that induce self-cleavage. In addition, we sought to increase the number of variant ribozymes of this class to further examine the proposed secondary structure model. Although numerous variant ribozymes were obtained that efficiently self-cleave, none exhibited changes in target specificity. These findings are consistent with the hypothesis that GlcN6P is used by the ribozyme as a coenzyme for RNA cleavage, rather than an allosteric effector.
Link
Kristian H
KH
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
Guo
Lixia
L
Breaker
Ronald R
RR
D001409:Bacillus cereus / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins; D008024:Ligands; D016337:RNA, Catalytic; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine