SUMMARY Ribosome-targeting antibiotics are indispensable both as therapeutic agents and as tools for basic research. In spite of the importance of these inhibitors, there are significant gaps in our understanding of the most fundamental principles of their action. Most of them interfere with protein synthesis by blocking the functional centers of the ribosome. Out of several functional centers, the catalytic peptidyl transferase center (PTC) and the nascent peptide exit tunnel (NPET) are the sites targeted by the broadest array of inhibitors. In the proposed project, we will explore the molecular mechanisms of action of the most basic PTC-targeting antibiotics and macrolides – chloramphenicol (CHL) and erythromycin (ERY). Recent studies yielded the unexpected conclusion that, in contrast to the general view of CHL and ERY as global and indiscriminate inhibitors, these antibiotics interfere with translation in a context-specific manner indicating that our understanding of their mechanism of action is incomplete and possibly even wrong. One way to obtain a clear explanation for the paradigm-shifting phenomenon of context-specific activity of PTC-acting inhibitors and macrolides is to directly visualize them within the ribosome complexes conducive to their action. Previous crystal structures uncovered how CHL and ERY bind to the PTC and NPET of the vacant bacterial ribosome and therefore provide information that is irrelevant for their context-specific activity. By determining the structures of CHL and ERY (as well as other PTC- acting drugs and macrolides) in functionally relevant ribosome complexes containing A-site aminoacyl-tRNA and P-site peptidyl-tRNA we will provide atomic-level view of their interactions not only with the ribosome (as before) but also with the growing peptide. Moreover, such structures could also reveal rearrangements that take place in the PTC of the ribosome upon drug binding and result in allosteric effects. Hence, in the Specific Aim 1, we will focus on obtaining the structures of 70S complexes carrying various aminoacyl-tRNAs in the A site in the presence and absence of CHL. Then, in the Specific Aim 2, we will obtain the first set of CHL-bound ribosome structures featuring dipeptidyl-tRNAs in the P site containing alanine, serine, or threonine in the penultimate position (the only sequence requirement for the efficient CHL-induced stalling). Finally, in the Specific Aim 3, we will provide structural and mechanistic insights into the context-specific activity of ERY and other macrolides. Once our proposed methodology is established and refined, we will expand it onto the newest FDA-approved clinically important drugs, such as linezolid, tedizolid, telithromycin, and solithromycin. The anticipated findings should significantly expand our understanding of the general mode of action of basic, as well as clinically- important, antibacterial drugs that act upon the catalytic center of the ribosome and may open new venues for...