Juliane K. Soukup

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. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.

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. Metal ions are also essential within the glmS ribozyme core for both structure and function of the ribozyme. Although metal ions do not directly promote catalysis, we show that metal ion identity and the varying physicochemical properties of metal ions have an impact on the rate of glmS ribozyme self‐cleavage. Specifically, these studies demonstrate that metal ion identity influences the overall apparent pKa of ribozyme self‐cleavage, and metal ion binding largely reflects phosphate oxygen affinity. Results suggest that metal ions take alternative roles in supporting the mechanism of catalysis.

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. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereusglmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the “sterically true” phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

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. In addition, it is the first riboswitch identified to utilize self-cleavage as a mode of genetic regulation in metabolism. Significant biochemical and biophysical data exist for the glmS ribozyme and aid in mechanistically understanding the importance of RNA and coenzyme structure to function in acid-base catalysis.

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). These techniques have been previously used to identify important functional groups in and tertiary contacts necessary for self-splicing and self-cleaving by catalytic RNAs, RNA-protein complexes, RNA folding, and RNA-metal ion interactions. Described here are the details of NAIM and NAIS experiments we have utilized to investigate RNA-ligand interactions between the glmS riboswitch and GlcN6P. These techniques can be employed to study a wide variety of RNA-small molecule interactions.