Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest.

The structural and functional uniqueness of the glmS ribozyme.

The glmS bacterial ribozyme/riboswitch is found in a number of Gram-positive bacteria, many of which are human pathogens. Investigation of the structure and function of the glmS catalyst will aid in the development of artificial agonists/antagonists that might function as novel antibiotics. The glmS ribozyme is mechanistically unique in that it is the first RNA catalyst identified to require a coenzyme, glucosamine-6-phosphate, for RNA self-cleavage.

Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding.

The bacterial glmS ribozyme is mechanistically unique among both riboswitches and RNA catalysts. Its self-cleavage activity is the basis of riboswitch regulation of glucosamine-6-phosphate (GlcN6P) production, and catalysis requires GlcN6P as a coenzyme. Previous work has shown that the coenzyme amine of GlcN6P is essential for glmS ribozyme self-cleavage, as is its protonation state.

Identification of metabolite-riboswitch interactions using nucleotide analog interference mapping and suppression.

Riboswitches are RNA elements capable of modulating gene expression through interaction with cellular metabolites. One member of the riboswitch family, the glmS riboswitch, is unique among riboswitches in that it modulates gene expression by undergoing self-cleavage in the presence of its metabolite glucosamine-6-phosphate (GlcN6P). In order to investigate the interactions between the glmS RNA and GlcN6P we performed nucleotide analog interference mapping (NAIM) and suppression (NAIS).

Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme.

The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function.

Ligand requirements for glmS ribozyme self-cleavage.

Natural RNA catalysts (ribozymes) perform essential reactions in biological RNA processing and protein synthesis, whereby catalysis is intrinsic to RNA structure alone or in combination with metal ion cofactors. The recently discovered glmS ribozyme is unique in that it functions as a glucosamine-6-phosphate (GlcN6P)-dependent catalyst believed to enable "riboswitch" regulation of amino-sugar biosynthesis in certain prokaryotes. However, it is unclear whether GlcN6P functions as an effector or coenzyme to promote ribozyme self-cleavage.

Riboswitches exert genetic control through metabolite-induced conformational change.

Conserved RNA structures have traditionally been thought of as potential binding sites for protein factors and consequently are regarded as fulfilling relatively passive albeit important roles in cellular processes. With the discovery of riboswitches, RNA no longer takes a backseat to protein when it comes to affecting gene expression. Riboswitches bind directly to cellular metabolites with exceptional specificity and affinity, and exert control over gene expression through ligand-induced conformational changes in RNA structure.

Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping.

The A-minor motifs appear to be the most ubiquitous helix packing elements within RNA tertiary structures. These motifs have been identified throughout the ribosome and almost every other tertiary-folded RNA for which structural information is available. These motifs utilize the packing of the donor adenosine's N1, N3, and/or 2'-OH against the 2'-OHs and minor groove edge of the acceptor base pair.

A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active.