Fluorescent Ligand Equilibrium Displacement: A High-Throughput Method for Identification of FMN Riboswitch-Binding Small Molecules.

Antibiotic resistance remains a pressing global concern, with most antibiotics targeting the bacterial ribosome or a limited range of proteins. One class of underexplored antibiotic targets is bacterial riboswitches, structured RNA elements that regulate key biosynthetic pathways by binding a specific ligand. We developed a methodology termed Fluorescent Ligand Equilibrium Displacement (FLED) to rapidly discover small molecules that bind the flavin mononucleotide (FMN) riboswitch. FLED leverages intrinsically fluorescent FMN and the quenching effect on RNA binding to create a label-free, in vitro method to identify compounds that can bind the apo population of riboswitch in a system at equilibrium. The response difference between known riboswitch ligands and controls demonstrates the robustness of the method for high-throughput screening. An existing drug discovery library that was screened using FLED resulted in a final hit rate of 0.67%. The concentration response of each hit was determined and revealed a variety of approximate effective concentration values. Our preliminary screening data support the use of FLED to identify small molecules for medicinal chemistry development as FMN riboswitch-targeted antibiotic compounds. This robust, label-free, and cell-free method offers a strong alternative to other riboswitch screening methods and can be adapted to a variety of laboratory setups.

Bacterial RNA-free RNase P: Structural and functional characterization of multiple oligomeric forms of a minimal protein-only ribonuclease P.

tRNAs are typically transcribed with extended 5' and 3' ends that must be removed before they attain their active form. One of the first steps of tRNA processing in nearly every organism is the removal of the 5' leader sequence by ribonuclease P (RNase P). Here, we investigate a recently discovered class of RNase P enzymes, Homologs of Aquifex RNase P (HARPs). In contrast to other RNase Ps, HARPs consist only of a metallonuclease domain and lack the canonical substrate recognition domain essential in other classes of proteinaceous RNase P. We determined the cryo-EM structure of Aquifex aeolicus HARP (Aq880) and two crystal structures of Hydrogenobacter thermophilus HARP (Hth1307) to reveal that both enzymes form large ring-like assemblies: a dodecamer in Aq880 and a tetradecamer in Hth1307. In both oligomers, the enzyme active site is 42 Å away from a positively charged helical region, as seen in other protein-only RNase P enzymes, which likely serves to recognize and bind the elbow region of the pre-tRNA substrate. In addition, we use native mass spectrometry to confirm and characterize the previously unreported tetradecamer state. Notably, we find that multiple oligomeric states of Hth1307 are able to cleave pre-tRNAs. Furthermore, our single-turnover kinetic studies indicate that Hth1307 cleaves pre-tRNAs from multiple species with a preference for native substrates. These data provide a closer look at the nuanced similarities and differences in tRNA processing across disparate classes of RNase P.