ABS 034: Cut to the Chase: Decoding The Kinetic Mechanism and Specificity of YloC in Bacillus subtilis

Chloe Caven ¹, Nupur Sehgal ², Michael E. Harris ³

¹ Division of Chemical Biology
² University of Florida
³

The Van Wickle Journal (2026) Volume 2, ABS034

Introduction: YloC from Bacillus subtilis, is representative of the novel class of YicC family enzymes. Their unique encapsulation mechanism sets this family of enzymes apart, as their substrate recognition relies on an unusual clamshell architecture that encapsulates native RNA substrates within an internal cavity. Despite emerging structural insight across the YicC family of endonucleases, the kinetic mechanism and YloC’s specificity remain unclear. This work applies a systematic approach to defining RNase specificity by integrating enzyme kinetics with structural and biochemical analyses. Building on known structures of YicC family endonucleases, we investigate how conformational changes regulate RNA recognition and catalysis. Here, we outline the kinetic mechanism of YloC to provide a quantitative basis for specificity and define some recognition including YloC’s substrate-size constraints and mapping cleavage-site distributions from gels that show multiple cut positions. Findings suggest that YloC exhibits broad substrate processing rather than strict single-target cleavage. The enzyme cleaves multiple RNA substrates, but specificity emerges from a combination of RNA structure and sequence. By establishing these general principles linking RNA structure, enzyme dynamics, and catalytic efficiency, this research advances a mechanistic understanding of bacterial RNase specificity. Together, these findings will aid the development of drugs targeting these enzymes in pathogenic species.

Methods: Multiple-turnover and single-turnover kinetic experiments were performed using fluorescently labeled RNA substrates to measure catalytic rates and evaluate substrate processing behavior. Reaction progress was monitored under varying substrate concentrations, pH conditions, and ion concentrations to determine factors influencing enzymatic activity.
Electrophoretic mobility shift assays (EMSA) were used to assess RNA binding affinity and substrate recognition. Cleavage products were resolved using denaturing gel electrophoresis, allowing visualization of cleavage-site distributions and substrate preferences. Comparative analyses between structured and bulged RNA substrates were conducted to evaluate the influence of RNA architecture on catalysis. Data obtained from kinetic fitting and gel analysis were integrated to construct a mechanistic model describing YloC substrate recognition and turnover behavior.

Results: YloC demonstrated broad substrate processing activity across multiple RNA substrates and conditions. Multiple-turnover kinetics showed efficient catalytic cycling, while single-turnover experiments revealed distinct fast and slow kinetic phases. EMSA analysis confirmed stable RNA binding and suggested that substrate recognition is influenced by RNA structure and local sequence composition. Cleavage-site mapping indicated that YloC does not rely on a single strict cleavage position but instead processes RNA at multiple accessible sites. Variations in pH and metal ion conditions altered catalytic efficiency, supporting the idea that conformational dynamics contribute to substrate recognition and catalysis.

Discussion: This study provides mechanistic insight into how YloC-family RNases recognize and process RNA substrates. Our findings support a model in which substrate specificity emerges from the combined effects of RNA structure, sequence, and enzyme conformational dynamics rather than rigid sequence recognition alone. The observed flexibility in substrate processing may allow YloC to participate in diverse RNA regulatory pathways within bacterial cells. These results advance the understanding of bacterial RNase specificity and establish a framework for future structural and mechanistic studies. Ultimately, YloC-family enzymes may represent promising targets for antimicrobial development in pathogenic bacterial species.