Project Summary The goal of this research project is to gain deeper mechanistic insights into trans-translation, a conserved bacterial system for translation quality control, directed proteolysis, and nonstop mRNA decay. The fundamental premise of our proposed studies is that the SmpB-tmRNA mediated trans-translation process solves all of problems caused by nonstop mRNAs, including rescue and recycling of unproductively stalled ribosomes, proteolysis of the potentially toxic nascent polypeptides, and selective decay of the causative defect mRNA. We propose that the tmRNA-rescued ribosome serves as a hub for recruitment of specialized rescue factors and initiation of interconnected salvage pathways. Therefore, over the next five years we will address two independent aspects of the trans-translation process: (1) adaptor guided proteolysis of tmRNA tagged proteins and (2) tmRNA-facilitated nonstop mRNA decay. In Aim I, we will investigate whether the translation machinery serves as a platform for initiating guided proteolysis by recruiting the AAA+ ClpXP protease system to translating ribosomes. The primary aim of our studies is to investigate a novel substrate recognition mechanism that enables the ClpXP protease, via its specific-enhancing factor SspB, to capture marked proteins at their site of biogenesis on the ribosome. A detailed knowledge of how specific substrate are captured via this novel pathway will shed significant new light on how AAA+ enzymes are directed to define cellular locations and how proteases contribute to cellular fitness and survival under adverse conditions. In Aim II, we will investigate the link between the tmRNA-mediated ribosome rescue system and the selective capture and decay of defective mRNAs by RNase R. We wish to elucidate the mechanism by which RNase R is recruited to the translation machinery and define its exact binding site and interacting partners on the rescued ribosome. We will explore the possibility that a unique modification of ribosomal components creates specialized ribosomes that play a key role in recruiting RNase R to tmRNA-rescued ribosomes. Recent studies have provided compelling evidence to demonstrate that the trans-translation process and ClpXP and Lon proteases are key participants in various regulatory pathways in several pathogenic bacteria, and therefore are required for pathogenesis. The genetic, biochemical, and structural studies proposed in this project offer the unique opportunity to gain significant new insights into the trans-translation process and identify new targets for future development of new antibiotics.