Understanding Mechanisms of Antibiotic Inhibition of Ribosomal Subunit Joining

Project Summary / Abstract Antimicrobial resistance is rapidly on the rise due to the excessive use and over-prescription of antibiotics in agriculture and medicine. Consequently, we are approaching a critical junction where bacterial infections may no longer be treatable with conventional antibiotics. To tackle this challenge, it is crucial to gain a comprehensive

understanding of the mechanism of action of conventional antibiotics. This understanding will serve as a valuable foundation for identifying strategies to rationally design novel antibiotics that emulate their mechanisms of action. This research project focuses on the bacterial ribosome, the essential cellular machinery responsible for protein

synthesis, as an antibiotic target. Specifically, this research will investigate the process of ribosome formation, which involves the assembly of large and small ribosomal subunits into a fully functional ribosome complex through a process called subunit joining. This process is targeted by various antibiotics, including orthosomycins,

pactamycin, and tetracyclines. Currently, the biophysical parameters governing the molecular mechanism of subunit joining and how antibiotics modulate these parameters to impede subunit joining remain largely unknown. This research will characterize the kinetic, thermodynamic, and structural dynamics underlying the

molecular mechanism of subunit joining and elucidate how antibiotics impact these parameters. The project comprises three specific aims, designed to actively engage undergraduate students and provide them with opportunities to contribute to scientific presentations and publications. In Aim 1 the thermodynamic parameters

that govern subunit joining will be measured by conducting pre-steady state stopped-flow kinetic experiments at various temperatures. These experiments will provide valuable quantification of the transition states and activation energies required for subunit joining. By introducing antibiotics during these experiments, how they

increase activation barriers, transforming subunit joining into a translation bottleneck can be assessed. In Aim 2 molecular simulations will be implemented to investigate the structural dynamics of the intersubunit interface and characterize non-covalent interactions that stabilize the fully formed ribosome. This approach will provide critical

insights into the interactions that are crucial to subunit joining. Implementing this as a characterization pipeline we can include antibiotics in molecular simulations to assess how they destabilize intersubunit bridges at an atomic level. Lastly, in Aim 3 cryo-electron microscopy will be performed to determine the structures of

meclocycline and methacycline bound to ribosomes, complexes for which no structural data is available. The obtained structures can subsequently be incorporated into the characterization pipeline described in Aims 1 and 2, enhancing our understandings of how antibiotics impede subunit joining. Overall, this research project aims

to provide significant insights into the molecular mechanism of ribosomal subunit joining. By comprehending how antibiotics alter the biophysical parameters of subunit joining, we can inform the development of novel antibiotics that can be rationally designed to specifically target subunit joining to inhibit translation.