Physiology of ribosome rescue in bacteria

SUMMARY Ribosome rescue pathways are conserved throughout bacteria, but the reason these pathways are important for physiology is not understood. The long-term goal of this project is to understand the function of ribosome rescue pathways and to target these pathways for new antibiotics. The overall objective of the proposed project is to identify interactions among components of the translation machinery that are specifically required for ribosome rescue and under what conditions different ribosome rescue systems are required. The central hypothesis of this work is that specific interactions within the ribosome and between the ribosome and other translation factors are uniquely required for ribosome rescue and that alternative rescue systems are critical under environmental conditions that cause RNA damage. The rationale for pursuing the proposed research is that it will determine why ribosome rescue is conserved in bacteria and will enable development of new antibiotics. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the molecular interactions required for trans-translation, 2) determine how ArfT rescues ribosomes in conjunction with either RF1 or RF2, and 3) determine why alternative ribosome rescue systems are required. Published work and preliminary data have identified small molecule inhibitors of trans-translation, and work in the first funded period of this grant identified their molecular targets. Biochemical and mutational analyses will be used in Aim 1 to determine why these targets are important for trans-translation and how the targets are disrupted by inhibitor binding. We will used structural and biochemical experiments in Aim 2 to determine the mechanism of a new alternative ribosome rescue pathway, ArfT, that can recruit either RF1 or RF2 to non-stop ribosomes. Our preliminary data identified conditions where the alternative ribosome rescue factor ArfB is required in Caulobacter crescentus, even when trans-translation is functional. We will determine the molecular basis for the ArfB requirement and determine of other alternative ribosome rescue factors are required under similar challenges in other bacteria. The use of small molecule inhibitors for chemical biology experiments to probe ribosome rescue is highly innovative, and the work proposed here is significant because it will delineate the physiological requirement for ribosome rescue pathways in bacteria and identify how these pathways can be inhibited.