High‐Throughput‐Compatible Assay for glmS Riboswitch Metabolite Dependence

Abstract

The increasing appearance of antibiotic-resistant strains of microbial pathogens seriously threatens our ability to control most of the major diseases. Coping with this threat will require continuous efforts to identify, explore and validate novel targets for antibiotic intervention. With the recent discovery that many bacteria utilize certain RNA domains, namely riboswitches and metabolite-dependent ribozymes, to regulate gene expression, a completely unexplored potential target class for new antibiotics has emerged. A broad-scale exploration of these targets requires the development of assay formats that are compatible with high-throughput screening (HTS) for modulatory small molecules. Here we report a HTScompliant fluorescence-based assay format that allows direct monitoring of the cis-cleaving reaction catalyzed by the glmS ribozyme. The glmS ribozyme is a cis-cleaving catalytic riboswitch located in the 5’-UTR of bacterial mRNA that codes for glucosamine-6-phosphate synthetase. The ribozyme can be specifically activated for glmS–mRNA cleavage by the metabolite glucosamine-6-phosphate (GlcN6P), that is, the metabolic product of the glmS-encoded protein itself. This complex regulation thus relies on a feedback-inhibition mechanism that senses the presence of metabolites that serve as cell-wall precursors. The ribozyme exhibits remarkable sensitivity and specificity for GlcN6P; related metabolites, such as glucose, glucose-6-phosphate or glucosamine (GlcN), cannot activate it. On the other hand, if small molecules can be found that activate the ribozyme in an analogous fashion to GlcN6P, they are likely to exhibit antibiotic activity because they trigger destruction of the mRNA that encodes for a protein required for the synthesis of a bacterial cell-wall precursor molecule. The glmS element resides upstream of the monocistronic glmS gene in 18 Grampositive organisms, including severely pathogenic ones like Staphylococcus aureus. We used the glmS ribozyme from Bacillus subtilis to test its suitability for the development of a HTS-compatible assay as a prerequisite for the search for ribozyme-activating small molecules. Because the natural glmS ribozyme is cis-cleaving, we sought to monitor this reaction as a function of GlcN6P concentration by fluorescence polarization (FP). FP can quantify dynamic binding events by measuring the amount of depolarization after excitation of fluorescent molecules with polarized light. This relates to the molecular weight of the excited molecule: if its molecular weight is high, it rotates and tumbles more slowly in space, and FP is preserved. If it is small, rotation and tumbling occur faster, and FP is reduced. The FP phenomenon has been used for analyzing binding events involving nucleic acids, such as in DNA– or RNA–protein interactions, or DNA detection by strand-displacement amplification. To apply FP to the detection of distinct, metabolite-dependent activation states of a catalytic riboswitch, we used an 81-nucleotide minimal ribozyme variant (Figure 1A). Fluores-